Expression systems utilizing autolyzing fusion proteins and a novel reducing polypeptide

ABSTRACT

The present invention provides expression systems for exogenous polypeptides wherein the polypeptide is expressed as a fusion protein together with clover yellow virus Nuclear Inclusion a (NIa), the NIa component serving to autolyze the fusion protein after expression. This system can be used to express a novel polypeptide which we have designated KM31-7 protein and which is capable of reducing dichloroindophenol and reduced glutathione. This polypeptide is useful in the treatment of disorders caused by oxidative stress.

FIELD OF THE INVENTION

[0001] The present invention relates to polypeptide expression systemsrequiring cleavage of a precursor product, and to proteases for use insuch systems. The present invention further relates to a novelpolypeptide capable of reducing dichloroindophenol and oxidizedglutathione, DNA encoding the novel polypeptide, vectors containing suchDNA, hosts transformed with such vectors, and pharmaceuticalcompositions containing the polypeptide. In addition, the presentinvention also relates to monoclonal antibodies against thispolypeptide, and a process for isolating and purifying the polypeptideusing such an antibody.

PRIOR ART

[0002] The Potyviruses are a group of viruses each of which have asingle-stranded, RNA genome of approximately 10,000 bases and whichinfects plants such as the family Solanaceae. The Potyvirus genome ischaracterized by possessing one extremely long open reading frame, orORF, [Dougherty, W. G. and Hiebert, E. (1980), Virology 101: 466-474.;Allison, R. et al. (1986), Virology 154: 9-20]. In order to express theindividual proteins encoded within the ORF, the translated polyproteinis digested by two types of protease, both of which are also encodedwithin the ORF [Dougherty, W. G. and Carrington, J. C. (1988), Ann. Rev.Phytopath. 26: 23-143].

[0003] Tobacco etch virus (TEV) is a member of the Potyvirus family, andthis virus produces nuclear inclusions which can be stained with trypanblue in the infected cell. The nuclear inclusions apparently consist oftwo kinds of protein, one of which has proven to be a viral protease,and which has been designated Nuclear Inclusion a, or NIa [J. Virol.,61: 2540-2548 (1987)].

[0004] The Nuclear Inclusion a proteases of the Potyviruses recognizeand cleave a peptide sequence which includes one of Gln-Gly, Gln-Ser andGln-Ala, and it is believed that this sequence is hexameric and occursat the C-terminal end of the relevant NIa within the polyprotein.Cleavage is between the two residues making up the dimers shown above.

[0005] The complete genomic sequences of TEV and tobacco vein mottlingvirus (TVMV), another member of the Potyvirus family, have beendetermined, and homology searching has allowed the location of the NIa'sof these viruses within their respective genomes [Virology, 154: 9-20(1986); Nucleic Acid Res., 14: 5417-5430 (1986)].

[0006] Clover Yellow Vein Virus, or CYVV, is also a Potyvirus. So far,only the gene occurring at the 3′ end of the CYVV genome, together withthe coat protein it encodes, has been sequenced [Uyeda, I. et al.(1991), Intervirol. 32: 234-245]. The structure of NIa region of thegenome has not previously been elucidated, nor has the corresponding NIabeen isolated.

[0007] The production of exogenous proteins by expression systems can bestraightforward, using techniques well known in the art. However, thereare many polypeptides which cannot easily be expressed in an exogenoussystem. The problem may be that the polypeptide cannot be expressed inlarge amounts, and this cannot usually be corrected merely by placing aregulatory gene upstream. Alternatively, it may be thatpost-transcriptional events required to obtain the mature form do nottake place, or take place incorrectly.

[0008] For example, many eukaryotic polypeptides are initiallytranslated with an N-terminal methionine which is subsequently deletedto obtain the mature form. This processing cannot take place inprokaryotes, so that alternative means of obtaining expression have hadto be found. One such technique involves fusing the desired exogenousprotein with maltose-binding protein or glutathione S-transferase, forexample, purifying the expressed fusion protein and then cleaving with aprotease, such as Factor Xa, enterokinase, or thrombin. The maindrawback of this cumbersome method is that it requires two purifictionsteps, which results in a substantial loss of the end product.

[0009] U.S. Pat. No. 5,162,601 discloses the use of TEV protease in themanufacture of a polyprotein having linker sequences between each of theproteins it is desired to express, such as human tPA. However, thispatent only discloses the cloning of a multigene encoding thispolyprotein into a host. There is no disclosure of expression orpurification of the proteolytically cleaved end product.

[0010] Oxygen for metabolic energy is generally provided in the form ofoxidizing agents in the cellular environment. The activated form inwhich the oxygen is used is generally as a free radical, such assuperoxide (O₂ ⁻), peroxide (H₂O₂) or a hydroxy radical (OH⁻), all ofwhich are reduced to form water (H₂O) after use. Oxygen gas, itself, ishighly oxidizing, but the term “activated oxygen”, as used herein,relates to oxygen and oxygen-containing molecules which have greateroxidizing potential than atmospheric oxygen. The most potent form ofactivated oxygen is the free radical, which is a molecule or atom havingone or more unpaired electrons.

[0011] Free radicals are typically unstable and, if not properlycontrolled, can denature lipids, proteins and nucleic acids.Consequently, although activated oxygen is essential to life, it is alsoa potential health hazard, and must be very closely controlled. Even invanishingly small amounts, activated oxygen can cause disorders, due tohigh reactivity. As a result, living organisms are unable to surviveunless they are equipped with a defence mechanism against activatedoxygen.

[0012] In the cellular environment, the locations, amounts and times ofgeneration of activated oxygen must be carefully balanced against thecell's ability to neutralize the associated danger. This ability istypically provided by a defence mechanism that uses its own antioxidantsor antioxidation enzymes. In the context of the present invention, an“antioxidant” is the generic name for a naturally occurring substancewhich is able to prevent or inhibit the auto-oxidation of lipids, forexample. The term “antioxidation enzyme” is used generically for anenzyme which catalyzes a reaction which eliminates activated oxygen, theterm “antioxidative action” being construed accordingly.

[0013] Excessive amounts of activated oxygen are produced in a number ofabnormal circumstances, such as when a person is stressed, is takingdrugs, smokes, undergoes surgery, has an organ transplant or if hesuffers ischemia through a cerebral or myocardial infarction. Theselarge amounts are more than the control systems of the body caneliminate, so that the excess of activated oxygen can cause furtherdamage to the body, seriously impairing normal cells. The resulting,so-called oxidative stress is responsible for a great many diseaseconditions.

[0014] To take arteriosclerosis as an example, the occurrence of lowspecific gravity lipoproteins which have been oxidized by activatedoxygen is considered to be one of the causes of the disease [Steinberg,D. (1983,) Arteriosclerosis 3, 283-301]. Oxidative stress is alsoconsidered to be intimately involved with cause and effect in themechanisms associated with the occurrence, metabolic abnormalities andvascular complications of diabetes [Kondo, M. ed., “Approaches fromModern Medicine (4) Free Radicals”, Medical View Pub., pp. 138-146].

[0015] Activated oxygen is also implicated in other pathological statesand conditions, such as; ischemic disorders (reperfusion disorders,ischemic heart disease, cerebroischemia, ischemic enteritis and thelike), edema, vascular hyperpermeability, inflammation, gastric mucosadisorders, acute pancreatitis, Crohn's disease, ulcerative colitis,liver disorders, Paraquat's disease, pulmonary emphysema,chemocarcinogenesis, carcinogenic metastasis, adult respiratory distresssyndrome, disseminated intravascular coagulation (DIC), cataracts,premature retinopathy, auto-immune diseases, porphyremia, hemolyticdiseases, Mediterranean anemia, Parkinson's disease, Alzheimer'sdisease, epilepsy, ultraviolet radiation disorders, radioactivedisorders, frostbite and burns.

[0016] Several defence mechanisms exist both inside and outside cellsfor the sole purpose of eliminating activated oxygen generatedphysiologically.

[0017] Intracellularly, antioxidants and antioxidative enzymes, such asthose given below, are known to process and eliminate activated oxygen.For example, catalase is present in peroxisomes, and this enzyme reducesand removes hydrogen peroxide. Glutathione peroxidase occurs in thecytoplasm and the mitochondria, and this enzyme reduces and detoxifieshydrogen peroxide and lipid peroxides in the presence of reducedglutathione. Transferrin, ferritin and lactoferrin, for example, inhibitthe generation of activated oxygen by stabilizing iron ions, whileceruloplasmin performs a similar function in connection with copperions. In addition, superoxide dismutase, which is present in thecytoplasm and mitochondria, catalyzes the reduction of superoxides toform hydrogen peroxide, the hydrogen peroxide then being eliminated bycatalase, for example. In addition, vitamins C and E, reducedglutathione and other low molecular weight compounds are also capable ofreducing and eliminating activated oxygen.

[0018] On the other hand, such agents as extracellular superoxidedismutase, extracellular glutathione peroxidase and reduced glutathioneexist in the extracellular environment, and these have similar modes ofaction to their intracellular counterparts listed above. However,compared to the intracellular situation, there are fewer types ofextracellular antioxidants and antioxidative enzymes, and there is onlya small number that exhibit extracellular antioxidative action.

[0019] Reduced glutathione has an important function in maintaining thereduced state both inside and outside cells, and it is represented bythe formula below. Glutathione was first discovered in yeast byde-Rey-Pailhade in 1888, and it was later named glutathione followingits isolation as a compound by Hopkins in 1921.

[0020] Glutathione is composed of three amino acids:—glutamic acid,cysteine and glycine. The thiol groups of two glutathione molecules canbe oxidized to form a disulfide bond in the presence of activatedoxygen, thereby reducing the activated oxygen.

[0021] Glutathione is mainly produced in the liver, and circulateswithin the body via the plasma. In the normal body, glutathione existsnearly entirely in the reduced form. When levels of the oxidized formincrease, then the reduced form is regenerated by the action ofglutathione reductase in the presence of nicotinamide adeninedinucleotide phosphate (NADPH). Thus, reduced glutathione protects thecell membrane from oxidative disorders and functions by the reducingactivated oxygen and free radicals. As a result of having thisantioxidative property, reduced glutathione also protects against theeffects of radioactivity and is useful as a therapeutic drug forcataracts. It has also recently been reported that systemic levels ofreduced glutathione are reduced in AIDS patients, which tends toindicate that the role of reduced glutathione in the body is extremelyimportant. However, in abnormal conditions, the amount of activatedoxygen can be so great that virtually all glutathione is in the oxidizedstate, so that activated oxygen is not removed as fast as possible.

[0022] Human thioredoxin is a second example of a substance which exertsvarious physiological actions by means of its reducing activity, bothinside and outside cells. Human thioredoxin (also known as Adult T CellLeukemia Derived Factor, ADF) was cloned as a factor which was capableof inducing interleukin 2 receptors (IL-2R) in adult T cell leukemiacell lines. It is a thiol-dependent reductase with two cysteine residuesat its active site, and it is capable of reducing activated oxygen andfree radicals.

[0023] In addition to inducing IL-2 receptors, human thioredoxin also:promotes cell growth in B cell strain 3B6 which is infected withEpstein-Barr virus (EBV); protects against tumor necrosis factor (TNF)derived from monocyte-origin cell line U937; and protects againstvascular endothelial cell impairment by neutrophils. Further, in theintracellular environment, human thioredoxin acts on the transcriptionfactors NFkB, JUN and FOS through its reducing activity, thereby topromote DNA bonding activity and enabling it to function to increasetranscriptional activity. Human thioredoxin is currently being developedas a protective agent for radioactivity disorders, as well as for atherapeutic drug for reperfusion disorders, rheumatoid arthritis andinflammations, all of which disorders can be protected against by itsability to protect against cellular impairment via its reducingactivity.

[0024] As has been described above, it is physiologically extremelyimportant to maintain both the intracellular and extracellularenvironments in a reduced state by elimination of activated oxygen andfree radicals. There are believed to be many, as yet unknown,antioxidants and antioxidative enzymes, both inside and outside cells,that have a role to play in removing activated oxygen and free radicals.Accordingly, it would be extremely useful to find a reducing substancewhich was capable of regenerating, for example, reduced glutathione.Such a substance could help in abnormal bodily conditions, such as thosedescribed above.

SUMMARY OF THE INVENTION

[0025] It is a first object of the present invention to provide a novelprotease, a nucleotide sequence encoding the protease, a vectorcontaining a DNA sequence encoding the protease, and a host cell whichhas been transformed with said vector.

[0026] It is a second object of the present invention to provide DNAencoding a protein of interest and also encoding the novel proteaseupstream from said protein, a sequence between the two said sequencesfurther encoding a peptide cleavable by said protease, all of saidsequences being in the same open reading frame. It is also an object toprovide a protein encoded by said DNA, as well as a vector containingsaid DNA and an expression system comprising said vector, said vectorbeing able to replicate autonomously in an appropriate host cell, suchas by comprising a nucleotide sequence required for autonomousreplication.

[0027] In the alternative, it is a first object of the present inventionto provide a nucleotide sequence encoding a novel polypeptide havingreducing activity in vivo. It is also an object to provide such DNAwhich encodes a peptide which is capable of reducing dichloroindophenoland oxidized glutathione.

[0028] It is a further object of the present invention to provide arecombinant vector comprising the above-mentioned DNA, said vector beingable to replicate autonomously in an appropriate host cell, such as byhaving a base sequence enabling autonomous replication.

[0029] Moreover, it is a yet further object of the present invention toprovide a host cell microorganism which has been transformed with theabove-mentioned recombinant vector. It is also an object to provide theabove-mentioned peptide as an expression product from the transformedhost cell, and to provide a monoclonal antibody against the peptide.

[0030] We have now identified and cloned the novel CYVV protease (NIa)and have surprisingly found that it is possible to use CYVV NIa as partof a fusion protein which can be expressed in such hosts as E. coli andwhich allows the production of large quantities of the fusion proteinwhich can self-cleave to yield the desired exogenous protein. The CYVVNIa gene can be stably maintained and expressed in Escherichia coli, andthe expressed NIa retains its activity as a specific protease, even whenthe protein forms part of a fusion protein.

[0031] We have also discovered DNA that codes for a novel polypeptidewhich is capable of reducing dichloroindophenol {also known asdichlorophenol-indophenol, 2,6-dichloroindophenol, or2,6-dichloro-4-[(4-hydroxyphenyl)imino]-2,5-cyclohexadien-1-one} andoxidized glutathione, said polypeptide being obtainable in large amountsby the use of appropriate genetic engineering techniques. Thispolypeptide is particularly useful in the therapy of conditions causedby, or related to, oxidative stress, or any disease caused by activatedoxygen, such as arteriosclerosis, diabetes and ischemic disorders(including reperfusion disorders, ischemic cardiac diseases,cerebroischemia and ischemic enteritis).

[0032] Thus, in a first aspect of the first embodiment of the presentinvention, there is provided a polynucleotide sequence wherein saidsequence comprises, in the 5′ to 3′ direction and in the same openreading frame:

[0033] a) a sequence encoding the clover yellow vein virus NuclearInclusion a protein, or a mutant or variant thereof having similarproteolytic specificity to that of clover yellow vein virus NuclearInclusion a protein;

[0034] b) a sequence encoding a peptide recognizable by and cleavable bysaid clover yellow vein virus Nuclear Inclusion a protein, or saidmutant or variant thereof; and

[0035] c) at least one sequence encoding a polypeptide.

[0036] The present invention also provides a sequence encoding theclover yellow vein virus Nuclear Inclusion a protein, or a mutant orvariant thereof having similar proteolytic specificity to that of cloveryellow vein virus Nuclear Inclusion a protein.

[0037] The present invention further provides a vector, especially anexpression vector, containing a sequence as defined above.

[0038] The present invention still further provides a host transformedwith a vector as defined above, as well as an expression systemcomprising said host and said expression vector, and also a polypeptideproduced by such an expression system.

[0039] In the alternative embodiment of the invention, there isprovided, in a first aspect, a polynucleotide sequence encoding apolypeptide having the amino acid sequence of amino acid numbers 1 to526 of sequence ID number 12, or which encodes a mutant or variant ofsaid polypeptide, provided that the polypeptide encoded by thepolynucleotide sequence is capable of reducing dichloroindophenol andoxidized glutathione.

[0040] There is also provided a vector, especially an expression vector,containing a sequence as defined above.

[0041] The present invention still further provides a host transformedwith a vector as defined above, as well as an expression systemcomprising said host and said expression vector, and also a polypeptideproduced by such an expression system.

[0042] The invention also provides the above polypeptide for use intherapy, as well as the use of such a polypeptide in the treatment andprophylaxis of conditions caused by, or related to, oxidative stress, orany disease caused by activated oxygen, and pharmaceutical compositionscomprising the polypeptide.

[0043] There is yet further provided an antibody, especially amonoclonal antibody, and equivalents thereof, against the polypeptide,and the invention additionally provides a method of producing such anantibody and a method of purification of the polypeptide using theantibody.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The present invention will be illustrated with respect to theaccompanying drawings, in which:

[0045]FIG. 1 is a restriction enzyme map of cDNA of the NIa regionisolated from CYVV-cDNA;

[0046]FIG. 2 shows construction of plasmid pKNI5′ containing a 5′-regionof NIa;

[0047]FIG. 3 shows construction of plasmid pKNI5IL containing a part ofIL-11 gene and a 5′-region of NIa;

[0048]FIG. 4 shows primers which were used to prepare the 5′IL DNAfragment the CIN3 DNA fragment and in which the 3′-terminus of NIa geneand the 5′-terminus of IL-11 gene are fused;

[0049]FIG. 5 shows the fusion of the CIN3 DNA fragment and the IL5′DNAfragment by PCR;

[0050]FIG. 6 shows the construction of plasmid pKSUN9;

[0051]FIG. 7 is a restriction enzyme map of pUCM31-7;

[0052]FIG. 8 is a comparative diagram of the nucleotide sequences of the3′ terminals in pUCKM31-7 and pcD-31;

[0053]FIG. 9 is a construction diagram of pSR α31-7;

[0054]FIG. 10 is a diagram of the introduction of a histidine hexamerencoding sequence into pUCTM31-7;

[0055]FIG. 11 is a construction diagram of pMAL31-7;

[0056]FIG. 12 is a diagram of the assay of dichlorophenol-indophenolreducing activity; and

[0057]FIG. 13 is a determination of oxidized glutathione reducingactivity.

DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention will be illustrated firstly by reference tothe first embodiment of the present invention, but the followingdiscussion is also appropriate to the second embodiment of theinvention, unless it is clear that the discussion is not applicable tothe second embodiment.

[0059] It will be appreciated that it is preferred for thepolynucleotide sequence of the invention generally to be in the form ofDNA, and references hereon in will generally be to DNA, for this reason.However, such references also include RNA, where appropriate. RNA is notso preferred, as uses therefor are limited by practicality. For example,mRNA can be expressed in xenopus oocytes or a wheatgerm lysate system,but neither of these is practical for producing large amounts of thefusion protein on an economic basis.

[0060] As used herein, the term “peptide” means any molecule comprising2 or more amino acids linked via a peptide bond. As such, the termincludes oligopeptides, polypeptides and proteins. The term “fusionprotein” relates to any single polypeptide obtained by combining two ormore other peptide sequences.

[0061] It will also be appreciated that the sequence of the invention ispreferred to be in the form of a double strand, and that the presentinvention also envisages the antisense sequence corresponding to thesequence of the invention. The double strand (or ds) sequence of theinvention may have one or two “sticky” ends, in which case the antisenseand sense strands will not necessarily correspond exactly.

[0062] The protein encoded by the sequence identified in a) above isintended to cleave the peptide encoded by the sequence of b) above inorder to release the polypeptide(s) encoded by the sequence of c) abovewhen the sequence of the invention is expressed in a suitable expressionsystem.

[0063] Cleavage of the fusion protein may take place at any time afterthe sequences of a) and b) above have been translated. As such, it willbe appreciated that the polypeptide encoded by c) above need not havebeen fully translated before cleavage. However, in practice, we havefound that at least some of the fusion protein, if not the majority, isfully translated prior to cleavage.

[0064] Where the sequence of c) above encodes more that one polypeptide,then it is necessary to encode further cleavage sequences between eachencoded polypeptide, unless it is desired, for example, to obtain anuncleaved, fused plurality of polypeptides.

[0065] If the sequence of c) above encodes more than one polypeptide,then the sequence may be susceptible to attenuation in a transcriptionenvironment. In the process of attenuation, transcription of the mRNAsequence of the invention stops before the whole of the mRNA has beenread, so that polypeptides encoded nearer the 3′ end of the sequencewill be produced in smaller amounts than those encoded nearer to the 5′end. Attenuation is not generally a problem, except where severalpolypeptides are encoded and/or the polypeptides are very long.

[0066] It is also generally preferred for the sequence of c) above toencode only one polypeptide, unless it is desired to prepare a pluralityof peptides for use together, or where such a plurality is fused.Otherwise, it is necessary to isolate the individual polypeptides aftercleavage, and this can be cumbersome and waste the end product throughpurification procedures.

[0067] However, where the sequence in c) above encodes more than onepolypeptide, and there is a cleavage sequence between each encodedpolypeptide, then the cleavage sequence does not necessarily have torecognized by CYVV NIa. All that is necessary is for the cleavagesequence between the sequence of a) and the 5′ end of the sequence of b)to be recognized by the protease encoded by the sequence of a). Where itis desired or acceptable to allow the fusion protein to self-digest toprepare a plurality of polypeptides, then any further cleavage sequencesmay be recognizable by the protease encoded by the sequence of a).However, such further sequences may be selected so that they arecleavable by other means, so that the fusion protein is cleaved by itsNIa portion after transcription to yield NIa and a polyprotein. The NIacan then be removed and the polyprotein cleaved by, for example, FactorXa or trypsin.

[0068] The cleavage peptide encoded by the sequence of b) above (alsoreferred to herein as “cleavage sequence” and “cleavage peptide”) may bea sequence which is wholly or partly comprised in either of thesequences encoded for by the sequences of a) and c) (“NIa” or“protease”, and “polypeptide”, respectively). Thus, the N-terminal endof the cleavage peptide may also be included in the C-terminal sequenceof the protease, while the the C-terminal portion of the cleavagepeptide may be included in the N-terminal portion of the polypeptide. Insuch an instance, the cleavage peptide has no independent existence, andthe recognition site for the protease is made up from the C-terminal ofthe protease and the N-terminal of the polypeptide.

[0069] The cleavage peptide may also be included in only part of eitherthe protease or the polypeptide. In such an instance, the N-terminal ofthe cleavage peptide will normally be included in the C-terminal portionof the protease, and the N-terminal portion of the polypeptide willeither be linked directly to the cleavage sequence or there may be oneor more amino acid residues between the polypeptide and the linker.

[0070] Where there are one or more amino acid residues between thecleavage sequence and the protease and/or the cleavage sequence and thepolypeptide, then the number and nature of such residues should be suchas not to prevent the action of the protease. Excess amino acid residueson the N-terminal of the polypeptide will not generally be advantageouswhere such residues have to be removed in order to obtain the matureform of the polypeptide. It is generally preferred, where possible, andin the absence of contraindications, to engineer the cleavage peptide sothat the mature form of the desired protein is obtained on cleavage ofthe fusion protein. However, it is entirely possible to obtain a proteinhaving Gly, Ser or Ala attached to the N-terminal for example, and thesecan be removed by the action of a suitable aminopeptidase, if desired.

[0071] In some instances, it may be desirable to encode a prolinebetween the N-terminal of the polypeptide and the cleavage sequence.This allows cleavage of residues up to the proline residue by the actionof aminopeptidase P (3.4.11.9), for example, but not beyond. Instead,the proline residue can then be removed by the action of prolineiminopeptidase to yield the mature protein. This forms a preferredembodiment of the invention.

[0072] The sequence of a) encodes a protease that is capable of cleavingthe fusion protein encoded by the sequence of the invention. In thepresent invention, this protease is clover yellow vein virus NuclearInclusion a protease, or a mutant or variant thereof having similarproteolytic specificity to that of clover yellow vein virus NuclearInclusion a protease.

[0073] The clover yellow vein virus NIa protease is encoded bynucleotide numbers 10 to 1311 in sequence ID number 1 in the sequencelisting, while the primary sequence of NIa is given by amino acidnumbers 4 to 437 in sequence ID number 2 in the sequence listing. Thesesequences are novel, and form a part of the present invention, as domutants and variants thereof.

[0074] Nuclear Inclusion a has proteolytic activity which specificallyhydrolyzes the peptide bond between Gln-Ala, Gln-Gly or Gln-Ser in asubstrate peptide. In addition, we have also found that NIa can cleaveGln-Val. Thus, in contrast to other proteases of the Potyvirus family,we have found that CYVV NIa (references to NIa herein should be taken asmeaning CYVV NIa, unless otherwise specified) can cleave the sequenceAsnCysSerPheGlnX, wherein X is any amino acid residue, but especiallyGly, Ala, Val or Ser, and particularly Gly, Ala or Ser.

[0075] It will also be appreciated that peptides comprising the sequenceAsnCysSerPheGlnX can be cleaved by NIa, especially where such sequenceis sterically exposed to the action of NIa. Thus, the present inventionalso provides a system for the preparation of a polypeptide, wherein aprecursor form of the polypeptide containing the sequenceAsnCysSerPheGlnX is cleaved by NIa. This system may also comprise otherprocessing steps, as appropriate, either before, after orcontemporaneously with the cleavage by NIa.

[0076] Although we generally prefer to use a sequence encoding naturallyoccurring NIa (in accordance with Sequence ID 2), the present inventionequally contemplates the use of mutants or variants of NIa.

[0077] As stated, the protease should have the same or similarspecificity as that of naturally occurring NIa. In this respect, theprotease should generally share substantial sequence homology with aminoacid residues 4 to 437 in sequence ID number 2 except where it isapparent to one skilled in the art that substantial variation ispossible without changing the recognition sequence or reducing activitybelow useful levels.

[0078] In general, it will be appreciated that the activity of any givenprotein is dependent upon certain conserved regions of the molecule,while other regions have little importance associated with theirparticular sequence, and which may be virtually or completely redundant.Accordingly, as stated above, the present invention includes anyvariants and mutants on the sequence which still show a specificitysimilar to that of naturally occurring NIa. Such variants and mutantsinclude, for example, deletions, additions, insertions, inversions,repeats and type-substitutions (for example, substituting onehydrophilic residue for another, but not strongly hydrophilic forstrongly hydrophobic as a rule). Small changes will generally havelittle effect on activity, unless they are in an essential part of themolecule, and such changes may be as a side-product of geneticmanipulation, for example, when generating extra restriction sites, ifsuch is desired.

[0079] In general, there will not usually be any particular reason towant to change the structure of NIa, except in circumstances apparent tothose skilled in the art. Indeed, most mutations and variations toeither the amino acid or coding sequence will be as a result of theisolation of novel variations on the original wild-type virus.Nevertheless deliberate, and even accidental, modifications are notexcluded from the present invention, provided that the protease has thenecessary specificity and sufficient proteolytic activity.

[0080] As used herein, the term “adverse effect” means any effect onspecificity or activity which renders the protease significantly lesseffective than the naturally occurring NIa identified above, to theextent that activity is reduced below a useful level.

[0081] Many substitutions, additions, and the like may be effected, andthe only limitation is that activity not be adversely affected. Ingeneral, an adverse effect on activity is only likely if the 3-D(tertiary) structure of the NIa is seriously affected.

[0082] The term “mutants” is used herein with reference to deletions,additions, insertions, inversions and replacement of amino acid residuesin the sequence which do not adversely affect activity. The presentinvention further includes “variants”, this term being used in relationto naturally occurring CYVV NIa which corresponds closely to sequence ID2, but which varies therefrom in a manner to be expected within a largepopulation. Within this definition lie allelic variation and thosepeptides from other cultivars showing a similar type of activity andhaving a related sequence.

[0083] It will be appreciated that neither the NIa nor the protein ofthe second embodiment of the present invention needs to correspondcompletely to the sequences depicted in Sequence ID's 2 and 12respectively. The only requirement is that each has the desiredactivity, even if the polypeptide is only a fraction of the whole of thenatural sequence, or even a mutant or variant of such a fraction.

[0084] The genes of eukaryotes, such as the interferon gene, aregenerally considered to demonstrate polymorphism [c.f., Nishi, T. et al.(1985), J. Biochem. 97, 153-159]. This polymorphism results in somecases where one or more amino acids are substituted in a polypeptide, aswell as other cases where there are no changes in the amino acidsequence, despite substitution of the nucleotide sequence.

[0085] Thus, it will be appreciated that the polynucleotide codingsequence may also be modified in any manner desired, provided that thereis no adverse effect on protease activity. Spot mutations and otherchanges may be effected to add or delete restriction sites, for example,to otherwise assist in genetic manipulation/expression, or to enhance orotherwise conveniently to modify the NIa molecule.

[0086] The terms “mutant” and “variant” are also used herein withreference to the polynucleotide sequence, and such references should beconstrued in an appropriate manner, mutatis mutandis. It will beappreciated that, while a mutant or variant of a peptide sequence willalways be reflected in the coding nucleotide sequence, the reverse isnot necessarily true. Accordingly, it may be possible for the nucleotidesequence to be substantially changed (see discussion of degeneracy ofthe genetic code below), without affecting the peptide sequence in anyway. Such mutants and variants of the nucleotide sequence are within thescope of the invention.

[0087] For example, it has been established that the protein obtained byreplacing a cysteine codon in the interleukin 2 (IL-2) gene with aserine codon is still capable of expressing IL-2 activity [Wang, A. etal. (1984), Science 224: 1431-1433]. Therefore, as long as a sequenceencodes a naturally occurring or a synthetic protein having theappropriate NIa activity, then it is included within the presentinvention.

[0088] A gene encoding the NIa of the invention may easily bereverse-engineered by one skilled in the art from sequence ID 2.

[0089] It will be appreciated that any one given reverse-engineeredsequence will not necessarily hybridize well, or at all, with any givencomplementary sequence reverse-engineered from the same peptide, owingto the degeneracy of the genetic code. This is a factor common in thecalculations of those skilled in the art, and the degeneracy of anygiven sequence is frequently so broad as to make it extremely difficultto synthesise even a short complementary oligonucleotide sequence toserve as a probe for the naturally occurring oligonucleotide sequence.

[0090] The degeneracy of the code is such that, for example, there maybe 4, or more, possible codons for the most frequently occurring aminoacids. Accordingly, therefore, it can be seen that the number ofpossible coding sequences for any given peptide can increaseexponentially with the number of residues. As such, it will beappreciated that the number of possible coding sequences for the NIa ofthe invention may have six or more figures. However, it may be desirableto balance the G+C content according to the expression system concerned,and other factors such as codon frequency for the relevant expressionsystem should generally be taken into account.

[0091] As stated above, hybridization can be an unreliable indication ofsequence homology but, nevertheless, those sequences showing in excessof 50%, preferably 70% and more preferably 80% homology with sequence ID1 are generally preferred. In each case, it will be appreciated that aprotease, as defined, should be encoded.

[0092] The present invention also envisages the possible use of a leadersequence encoded upstream of the protease. This would allow the fusionprotein to be externalized from the host, and collected in the culturesupernatant, for example. The resulting externalized fusion proteinwould then be allowed to self-digest, and the polypeptide collected. Forsuch signal sequences, any suitable sequence may be used, especiallywhere such has been specifically developed for a given expressionsystem.

[0093] The present invention also envisages vectors containing thesequence of the present invention. The general nature of vectors for usein accordance with the present invention is not crucial to theinvention. In general, suitable vectors and expression vectors andconstructions therefor will be apparent to those skilled in the art, andwill be chosen according to precisely what the practitioner wishes toachieve with the sequence, such as cloning or expression.

[0094] Suitable expression vectors may be based on ‘phages or plasmids,both of which are generally host-specific, although these can often beengineered for other hosts. Other suitable vectors include cosmids andretroviruses, and any other vehicles, which may or may not be specificfor a given system. Suitable control sequences, such as recognition,promoter, operator, inducer, terminator and other sequences essential toand/or useful in the regulation of expression, will be readily apparentto those skilled in the art, and may be those associated with CYVV orwith the vector used, or may be derived from any other source assuitable. The vectors may be modified or engineered in any suitablemanner.

[0095] It will be appreciated that sequence ID 2 represents sufficientsequence to encode entire NIa. Terminators, promoters and other suchcontrol sequences as desired may be added so as to, for example,facilitate ligation into a suitable vector, or expression, or both.

[0096] It will be appreciated that a DNA fragment encoding the NIa ofthe invention, together with any fragment encoding the cleavage sequenceand that encoding the polypeptide(s) may easily be inserted into anysuitable vector. Ideally, the receiving vector has suitable restrictionsites for ease of insertion, but blunt-end ligation, for example, mayalso be used, although this may lead to uncertainty over the openreading frame and direction of insertion. In such an instance, it is amatter of course to test hosts transformed with the transfected vectorto select vectors having the necessary fragments inserted in the correctdirection and in the correct ORF. In order to ensure that the fragmentsare in the correct ORF, it may be desirable to create a construct of thesequences a), b) and c) which can then be inserted directly into thevector, thereby reducing the uncertainty of obtaining the desiredexpression vector. Suitable vectors may be selected as a matter ofcourse by those skilled in the art according to the expression systemdesired.

[0097] By transforming E. coli, for example, with the plasmid obtained,selecting the transformant with ampicillin or by other suitable means,and adding tryptophan or other suitable promoter inducer (such asindoleacrylic acid), the desired fusion protein may be expressed. Theextent of expression may be analyzed by SDS polyacrylamide gelelectrophoresis—SDS-PAGE [Laemmli et al., Nature, (1970), 227,pp.680-685].

[0098] It will also be appreciated that, where another vector is used,for example, it will be equally acceptable to employ any suitableselection marker or markers, or an alternative method of selection,and/or to use any suitable promoter as required or convenient.

[0099] After cultivation, if the fusion protein is to be collected fromthe host cells, then the transformant cells are suitably collected,disrupted, for example sonicated, and spun-down. Disruption may also beby such techniques as enzymic digestion, using for example cellulase, orby shaking with an agent such as glass beads, but methods such assonication are generally preferred, as no extra ingredients arenecessary. The resulting supernatant may be assayed for polypeptideactivity and the cleavage products can be determined by SDS-PAGE, forexample.

[0100] Conventional protein purification is suitable to obtain theexpression product.

[0101] The DNA of the present invention may be prepared by isolating theRNA genome from clover yellow vein virus [Uyeda, I. et al. (1975) Ann.Phytopath. Soc. Japan 41: 92-203]. A suitable source of CYVV is AmericanType Culture Collection No. PV 123. In any event, clover yellow veinvirus may be defined as a virus which causes necrosis in Vicia faba.

[0102] Genomic RNA may be obtained from CYVV particles which have beenpurified from an infected plant and then reverse transcribed anddouble-stranded cDNA prepared by known methods.

[0103] For the purposes of determining whether a virus is a mutant orvariant of the same strain of CYVV, sequence homology is a goodindicator. Many types of Potyvirus are known, and they all vary inpathogenicity. Whether a given virus forms a separate member of thefamily is based on the serological relation of the viral coat proteinsand on the homology of the amino acid sequences. Accordingly, viruseswhich have primary amino acid sequences which share 90% homology areconsidered to be the same strain, while viruses which have primary aminoacid sequences which share less than 70% homology are considered todistinct family members [Shukla, D. D. and Ward, C. W. (1989), Arch.Virol. 106: 171-200]. Based on the various properties of the coatprotein of CYVV used in the present invention [Uyeda, I. et al. (1991),Intervirol. 32: 234-245], CYVV is recognized as an independent member ofthe Potyvirus family. Therefore, any virus having a coat protein primaryamino acid sequence homology of 90% or more, or any virus detecting aspositive by ELISA using an anti-CYVV anti-serum (for example, AmericanType Culture Collection No. PVAS-123: clover yellow vein virusantiserum), is defined as being a strain of clover yellow vein virus.

[0104] The various plant species upon which CYVV can be grown include;Phaseolus vulgaris, Vicia faba and Pisum sativtum, but Vicia faba,especially Vicia faba cultivar Wase-soramame, is preferred.

[0105] The preferred method for purifying the virus particle involveshomogenizing and squeezing a leaf or leaves of an infected plant in asuitable buffer, followed by extraction with an organic solvent, such aschloroform, and repeating differential centrifugation, followed bysucrose density gradient centrifugation.

[0106] One way to confirm that the virus particle thus obtained is CYVVcan be performed by examining the virus particle under an electronmicroscope. Another is by inoculating Vicia faba with the virus particlein order to observe whether any symptoms occur.

[0107] Suitable methods for extracting the genomic RNA from virusparticles include the guanidinium thiocyanate/phenol method, theguanidinium thiocyanate/trifluoro-cesium method and the phenol/SDSmethod. However, we prefer to use the alkaline sucrose density gradientcentrifugation method [Dougherty, W. G. and Hiebert, E. (1980) Virology101: 466-474].

[0108] The RNA obtained as described above can then be tested to confirmthat it indeed encodes a protease by translation in a cell-freetranslation system. Autolysis (self-digestion) can then be detectedwhere the RNA codes for more than just the protease in the totaltranslation product, by monitoring any change in the molecular weightsof harvested products from the lysates. Such monitoring can be performedby means of an anti-coat protein antibody, for example.

[0109] Should it be required, then the production of translation productcan be monitored with passage of time using an anti-coat proteinantibody, for example, with the genomic RNA being translated byinjection into Xenopus laevis oocytes [Gurdon, J. B. (1972), Nature 233:177-182], or by using a rabbit reticulocyte or a wheat germ lysatesystem [Schleif, R. F. and Wensink, P. C. (1981), in “Practical Methodsin Molecular Biology” Springer-Verlag, N.Y.].

[0110] Single stranded DNA can be synthesized by using the thus obtainedgenomic RNA as a template using a reverse transcriptase, and ds-cDNA canbe synthesized from the single-stranded (ss) cDNA by following standardprocedures. Suitable methods include the S1 nuclease method[Efstratidiatis, A. et al. (1976), Cell 7: 279-288: Okayama, H. andBerg, P. (1982), Mol. Cell. Biol. 2: 161-170 and others], the Landmethod [Land, H. et al. (1981) Nucleic Acids Res. 9, 2251-2266] and theO. Joon Yoo method [Yoo, O. J. et al. (1983) Proc. Natl. Acad. Sci. USA79, 1049-1053]. Of the various options, we prefer to use theGubler-Hoffman method [Gubler, U. and Hoffman, B. J. (1983), Gene 25:263-269].

[0111] The thus obtained ds-cDNA can then be incorporated into a cloningvector, such as a plasmid or lamdha phage, and the resulting recombinantvector then transformed into a microorganism, such as Escherichia coli.E. coli strain DH5α is particularly preferred. Transformants can then beselected by their resistance to bactericidal agents, such astetracycline or ampicillin by techniques well known in the art.

[0112] Transformation can, for example, be carried out by the Hanahanmethod [Hanahan, D. (1983), J. Mol. Biol. 166: 557-580], wherein therecombinant DNA vector is introduced into a cell which has been madecompetent by treatment with calcium chloride, magnesium chloride orrubidium chloride.

[0113] Suitable methods for selecting transformants having NIa DNA areas shown below.

[0114] (1) Screening with a Probe

[0115] If it is desired to start from wild-type CYVV, then one way toisolate the appropriate RNA is to use a cDNA probe, given that the aminoacid sequence of NIa has been elucidated (the portion of the sequenceused may be from any region of NIa). Thus, an oligonucleotidecorresponding to the relevant amino acid sequence is synthesized. Ingeneral, the amino acid sequence chosen will involve the least amount ofdegeneracy possible, otherwise it will be necessary to produce severalprobes using the various codons possible. In such an instance, it islikely to be of assistance to take codon usage frequency intoconsideration. Alternatively, plural nucleotide sequences can beconsidered, and inosine can be used to replace nucleotides which vary.The probe can then be labeled with a radioisotope, such as ³²P, ³⁵S orbiotin. Transformant strains can then be detected by fixing denaturedplasmid DNA on nitrocellulose filters using the radiolabelled probes,positive clones being detectable by autoradiography.

[0116] (2) Using a PCR Probe

[0117] In this technique, oligonucleotides both from the sense strandand from the anti-sense strand corresponding to a portion of the knownamino acid sequence can be synthesized and the polymerase chain reaction[Saiki, R. K. et al. (1988), Science 239, 487-491] carried out. Thesecan then be used in combination to amplify a DNA fragment encoding NIa.Suitable template DNA is cDNA obtained through reverse transcription ofviral genomic RNA known to encode NIa. The thus prepared DNA fragmentsare labeled, such as with ³²P, ³⁵S or biotin, and colony hybridizationor plaque hybridization is carried out with this probe to select theclone of interest.

[0118] (3) Screening by Exogenous Production in an Animal Cell

[0119] This method involves culturing a transformant strain to amplify agene, followed by transfecting the gene into an animal cell (generallyusing either a plasmid which is replication competent and containing atranscription promoter region, or using a plasmid which can beintegrated into an appropriate chromosome) to express a protein encodedby the gene. By measuring the relevant activity, a strain can beselected.

[0120] (4) Selection Using an Antibody Against NIa

[0121] Antibody is produced against the nuclear inclusions (NIa and NIb)from a plant infected with CYVV, or is produced against a proteinproduced by an expression vector in an appropriate system. The antibody,or its anti-antibody, can then detect the desired NIa or strain ofinterest.

[0122] (5) Selective Hybridization Translation System

[0123] Transformant cDNA is hybridized with mRNA from cells whichexpress NIa, as described above, and the mRNA bound to the cDNA isdissociated and recovered. The recovered mRNA is translated into proteinin a translation system, for example, by injection into Xenoous laevisoocytes, or into such cell-free systems as rabbit reticulocyte lysate orwheat germ lysate. The strain of interest is selected by examining theactivity of NIa or by detecting NIa by means of an antibody to NIa.

[0124] The DNA encoding CYVV NIa can be obtained from the transformantstrains of interest by known methods [c.f. Maniatis, T. et al. (1982),in “Molecular Cloning A Laboratory Manual” Cold Spring HarborLaboratory, N.Y.]. For example, a fraction which corresponds to a vectorDNA is separated from the cells, and the DNA region which codes for saidprotein is excised from said plasmid DNA.

[0125] Determination of the thus obtained DNA sequence can be carriedout by using, for example, the Maxam-Gilbert chemical modificationmethod [Maxam, A. M. and Gilbert, W. (1980), in “Methods in Enzymology”65: 499-2761, or by using, for example, a dideoxynucleotide chaintermination method using the M13 phage [Messing, J. and Vieira, J.(1982), Gene 19: 269-276].

[0126] In recent years, fluorochromes have tended to replace the use ofthe more dangerous radioisotope for the determination of DNA sequences.In addition, dideoxynucleotide chain termination is now generallyperformed by a robot under computer control. Systems which read basesequences after electrophoresis are also proliferating, and examplesinclude the “CATALYST 800” sequencing robot and the 373A DNA sequencer(Perkin-Elmer Japan Applied Biosystems). These systems enable DNA basesequence determination procedures to be performed both efficiently andsafely.

[0127] The vectors of the present invention can generally be soorganized that they can be expressed in “standard cells”, eitherprokaryotic or eukaryotic. In addition, by introducing an appropriatepromoter and a sequence for phenotypic expression into the vector, thegene can be expressed in assorted host cells.

[0128] Suitable prokaryotic hosts include Escherichia coli and Bacillussubtilis. For phenotypic expression of the relevant gene, the vector cansuitably contain a replicon originating from a species which iscompatible with the host. In E. coli, a plasmid might contain areplication origin and a promoter sequence such as lac or UV5. Vectorswhich can confer selectivity based on phenotypic character (phenotype)to a transformed cell are preferred.

[0129]Escherichia coli is often used as the host, and strain JM 109derived from E. coli strain K12 is a preferred host. Vectors for E. coliare, presently, generally selected from pBR322 or the pUC series ofplasmids, but other strains and vectors can be used, as desired.Suitable promoters for Escherichia coli include the lactose promoter(lac), the tryptophan promoter (trp), the tryptophan-lactose (tac)promoter, the lipoprotein (lpp) promoter, the lambda (λ) PL promoter(from λ phage) and the polypeptide chain elongation factor Tu (tufB)promoter, but the present invention is not limited to these promoters.

[0130] Suitable strains of Bacillus subtilis include strain 207-25.Suitable vectors include pTUB228 (Ohmura, K. et al. (1984), J. Biochem.95: 87-93] and others. A suitable promoter is the regulatory sequence ofthe Bacillus subtilis α-amylase gene. This is an appropriate examplewherein a signal peptide sequence (from α-amylase), can be used forextracellular secretion.

[0131] If it is desired to express the fusion protein in eukaryoticcells, then cells from vertebrates, insects, yeasts, plants, etc. may beused. The preferred vertebrate cells are COS cells, especially COS-1cells [e.g. Gluzman, Y. (1981), Cell 23: 175-182], or a Chinese hamsterovary cell line (CHO) deficient in dihydrofolate reductase [Urlaub, G.and Chasin, L. A. (1980), Proc. Natl. Acad. Sci. USA 77, 4216-4220],although the present invention is not limited to these.

[0132] As stated above, COS cells are suitably employed as vertebratehost cells, and these may be used as an example. Expression vectorscontaining an SV40 replicon are able to replicate autonomously in COScells, and these are provided with a transcription promoter, atranscription termination sequence and one or more splicing sites.Expression vectors containing the desired DNA sequence can be used totransform COS cells by various methods, such as, for example, theDEAE-dextran method [Luthman, H. and Magnusson, G. (1983), Nucleic AcidsRes. 11, 1295-1308], the calcium phosphate-DNA coprecipitation method[Graham, F. L. and van der Eb, A. J. (1973), Virology 52, 456-457] andthe electroporation method [Neumann, E. et al. (1982), EMBO J. 1,841-8451.

[0133] If CHO cells are used as the host cells, then it is appropriateto use a vector capable of expressing the neo gene which serves toprovide G418 resistance. Suitable vectors carrying this marker includepRSVneo [Sambrook, J. et al. (1989): “Molecular Cloning—A LaboratoryManual”, Cold Spring Harbor Laboratory, NY] and pSV2-neo [Southern, P.J. and Berg, P. (1982), J. Mol. Appl. Genet. 1, 327-341]. Transformantscan then be selected by their resistance to G418.

[0134] The selected transformant can be cultured by conventional methodsand, in the case of the second embodiment of the invention, polypeptideis produced both intracellularly and extracellularly. Suitable culturemedia can be selected from those commonly used, in accordance with thehost cells employed. For example, for COS cells, a medium containingblood components, such as fetal bovine serum, can be added as necessaryto media such as RPMI-1640 medium or Dulbecco's modified Eagle's medium(DMEM).

[0135] Should the practitioner prefer to use insect cells, then cellsderived from Spodoptera frugiverda [Smith, G. E. et al. (1983), Mol.Cell. Biol. 3: 2156-2165] may be used.

[0136] Suitable yeasts include baker's yeast (Saccharomyces cerevisiae)and fission yeast (Schizosaccharomyces pombe).

[0137] Suitable plant cells include those from Nicotiana tabacum andOryza sativa.

[0138] It will be appreciated that the hosts enumerated above arestandard hosts in the art, and that the skilled person in the art willbe able to choose amongst these and other hosts as appropriate.

[0139] Suitable expression vectors for vertebrate cells include thosewhich have a promotor located upstream from the gene to be expressed,together with such sites as an RNA splicing site, a polyadenylationsite, and a transcription-termination sequence, and further having areplication origin, if required. A suitable example of such anexpression vector is pSV2dhfr, which has the SV40 early promotor[Subramani, S. et al. (1981), Mol. Cell. Bio. 1: 854-864].

[0140] A suitable expression system for insect cells includes culturedcells of Spodoptera frugiperda. Suitable expression vectors have, forexample, the Baculovirus Polyhedrin promoter located upstream from thegene to be expressed, together with a polyadenylation site and a portionof the AcMNPV (Acutogranha californica nuclear polyhedrosis virus)genome required for homologous recombination. One example is pBacPAK8[Matuura, Y. et al. (1987), J. Gen. Virol. 68: 1233-1250].

[0141] For eukaryotic expression, yeast is commonly used, such asbaker's yeast (S. cerevisiae). Suitable expression vectors for yeast mayinclude the alcohol dehydrogenase promoter [Bennetzen, J. L. and Hall,B. D. (1982), J. Biol. Chem. 257: 3018-3025], the acidic phosphatasepromoter [Miyahara, A. et al. (1983), Proc. Natl. Acad. Sci. USA 80,1-5], or the carboxypeptidase Y promoter [Ichikawa, K. et al. (1993),Biosci. Biotech. Biochem. 57: 1686-1690], for example. In such aninstance, the signal peptide sequence from carboxypeptidase Y may alsobe used, in order to effect secretion to the extracellular space.

[0142] Suitable expression vectors for plants include, for example,pBI121 which has a ³⁵S promoter (derived from the early promoter ofcauliflower mosaic virus), the polyadenylation sequence of the nopalinesynthesis gene from Agrobacterium tumefaciens, and the Agrobacteriumtumefaciens gene transfer sequence [Jefferson, R. A. et al. (1987), EMBOJ. 6: 3901-39071. Such vectors can be introduced into plant cells bysuch methods as infection with Agrobacterium tumefaciens andelectroporation.

[0143] Plasmid pKK388-1 (manufactured by Clonetech Co.) has a trcpromoter and is suitable for use in Escherichia coli. This expressionvector is able to autonomously replicate in a strain derived fromEscherichia coli strain K12, such as strain JM109. This vector caneasily be introduced into Escherichia coli by such well known methods asare mentioned above. The thus obtained strain can be inoculated into amedium, such as the well known LB medium, and cultured for a while.

[0144] The trc promoter can then be activated by adding, for example,isopropyl-β-galactopyranoside (IPTG), to induce the promoter. Afterfurther culturing, the expressed protein then can be extracted from thecells by disrupting the cells, such as with a sonicator. If it isdesired to produce a protein having Pro at the N-terminus, then it willgenerally be appropriate to use Escherichia coli as the host.

[0145] By applying the above description and, if necessary, taking intoaccount the accompanying Examples, a fraction from the culture ofsuitably transformed cells containing the desired polypeptide can beisolated and purified by known methods, depending on the physical andchemical properties of the polypeptide. Suitable such methods includetreatment with a protein precipitant, ultrafiltration, variouschromatographies (such as molecular sieve chromatography (gelfiltration), adsorption chromatography, ion exchange chromatography,affinity chromatography and high performance liquid chromatography(HPLC)], dialysis and combinations of the above methods.

[0146] In order to determine whether the expressed NIa protein has thenecessary protease activity, the purified NIa protein may be reactedwith a substrate protein containing a cleavage sequence for theprotease, such as the expression product of the gene encoding the fusionprotein which includes the viral coat protein (the natural substrate ofNIa) together with nuclear inclusion b (NIb) (Dougherty, W. G. et al.(1988), EMBO J. 7: 1281-1287]. Such a fusion protein may be isolatedfrom the viral genomic cDNA and inserted into an expression vector. Theresulting expression vector is then introduced into a suitable host cellto produce the fusion protein. By treating the resultant fusion proteinin known manner, such as with a protein precipitant or bychromatography, the fusion protein can be separated and purified.

[0147] The thus separated and purified substrate protein can then bereacted with said protease in a suitable buffer solution at a suitabletemperature to recover a reaction product. The recovered reactionproduct can then be subjected to electrophoresis and to Western blottingusing an anti-coat protein antibody to allow the protease activity to bedetected as a difference in the mobility of a band. In this method, itis also possible for a a synthetic oligopeptide containing anappropriate cleavage sequence to be used.

[0148] The proteolytic activity of the protease of the present inventioncan be detected without purification. Thus, activity can be measured byconnecting the protease gene in sequence and in the same open readingframe, downstream from a promoter, such as the trc promoter, andupstream from a cleavage sequence for the protease. If the protease isactive in the expression product, then a band of higher mobility thanthe fusion protein will be observable by Western blotting.

[0149] By recovering the band from the gel in which the cleavage isobserved and by analyzing the amino terminal sequence thereof byconventional methodology, it can be established whether the protein iscleaved at the desired peptide bond.

[0150] The protein which it is desired to produce may be produced, forexample, as a fusion protein together with a protein such as glutathioneS-transferase, and the NIa of the present invention can then be used tocleave the linker sequence in vitro. In one alternative, the desiredprotein is directly expressed in a host cell as a fusion proteintogether with said protease and the desired protein is prepared byself-cleavage.

[0151] If desired, NIa can easily be produced in a high yield and highpurity using the above methods, and the thus obtained recombinant NIa ofthe present invention can be used as a protease.

[0152] If it is desired to chemically synthesize the DNA's of thepresent invention, then these can be prepared by conventionalmethodology, such as the phosphite triester method [Hunkapiller, M. etal. (1984), Nature 310: 105-111] or by the chemical synthesis of nucleicacids [Grantham, R. et al. (1981), Nucleic Acids Res. 9: r43-r74]. Ifdesired, partial modification of these nucleotide sequences can beeffected by conventional methods, such as by site-specific mutagenesis,using a primer comprising a synthetic oligonucleotide encoding thedesired modification [Mark, D. F. et al. (1984), Proc. Natl. Acad. Sci.USA. 81: 5662-5666].

[0153] Hybridization, as mentioned above, can be established, forexample, by using a probe labeled with [α-³²P]dCTP, for example, in therandom primer method [Feinberg, A. P. and Vogelstein, B. (1983) Anal.Biochem. 132: 6-13], or by nick translation [Maniatis, T. et al. (1982),in “Molecular Cloning A Laboratory Manual” Cold Spring HarborLaboratory, N.Y.]. The DNA is fixed to a solid phase by a conventionalmethod, for example by adsorbing to a nitrocellulose membrane or a nylonmembrane, and then heating or using ultraviolet radiation. The solidphase is then typically immersed in a prehybridization solutioncontaining 6×SSC, 5% Denhardt's solution and 0.1% SDS and incubated at55° C. for 4 hours or longer. Then, the previously prepared probe isadded to the prehybridization solution to a final specific activity of1×10⁶ cpm/ml, and the mixture incubated at 60° C. overnight. Then, thesolid phase is washed five times repeatedly with 6×SSC for 5 minutes atroom temperature, followed by washing at 57° C. for 20 minutes, andautoradiography is carried out to determine whether the DNA hashybridized or not. It will be appreciated that this is a specificexample, and that other methods are equally possible.

[0154] The desired protein may be prepared by either an intracellulardirect cutting method and an extracellular cutting method.

[0155] Intracellular Direct Cutting Method

[0156] DNA encoding NIa is connected with DNA encoding the desiredprotein via a cleavage sequence, preferably Gln-Gly, Gln-Ser or Gln-Ala.The resulting DNA encodes a fusion protein and is inserted into a vectorcomprising a promoter and a terminator and is used to obtain expressionin an appropriate host cell. The resulting, expressed fusion proteinself-cleaves through the protease activity, thereby affording a peptidein which Gly, Ser or Ala is attached by a peptide bond at the N-terminusof the protein of interest. A particularly preferred protein forexpression is IL-11.

[0157] Where it is desired to cleave any residues from the N-terminus ofthe resulting protein, then this may be done as described above, usingaminopeptidase P, for example, to leave an N-terminal Pro residue,should this be desired.

[0158] Some expression systems already have aminopeptidase P present.However, if aminopeptidase P is not present, and it is desired to cleaveN-terminal amino acid residues in situ, then an expression vector foraminopeptidase P may be introduced into the host cell. Aminopeptidase Pis a known protein, and a gene sequence for the enzyme is readilyavailable to those in the art.

[0159] When it is desired to obtain a protein which starts with Pro atthe N-terminus, then it is generally advantageous to extend cultureduration in order to allow the cellular aminopeptidase P to act.

[0160] As described above, the N-terminal Pro residue can be removed bythe catalytic action of proline iminopeptidase (3.4.11.5), endogenouslyexpressed or expressed by an exogenous expression vector (the enzyme isa known protein, and a gene sequence for the enzyme is readily availableto those in the art). Alternatively, the proline residue can be cleavedafter recovery from the expression system, such as where the fusionprotein is secreted from the cell.

[0161] Thus, it can be seen that the present invention permits theproduction of proteins having any desired N-terminal residue.

[0162] Where the sequence of the present invention results in apolypeptide having an Ala N-terminus, then this alanine residue can beremoved by the catalytic action of alanine aminopeptidase (3.4.11.14)which, again, is a known enzyme, and wherein the gene sequence isreadily available to those skilled in the art. This technique alsoallows the production of a polypeptide having a freely chosen N-terminalresidue.

[0163] The desired protein can then be isolated and purified by wellknown methods.

[0164] Extracellular Cutting Method

[0165] NIa can be produced by incorporation of suitable DNA into avector which is then included in a suitable expression system. Theresulting NIa expressed by the transformed cell can then purified by useof such as an ion exchange column, a gel filtration column or a reversephase column. Alternatively, the NIa may be expressed as a fusionprotein with another polypeptide, such as glutathione-S-transferase ormaltose-binding protein, which can be separated and purified using aglutathione column or a maltose column, respectively. After cutting thepurified product with enterokinase or Factor Xa, the NIa can be purifiedand used.

[0166] Meanwhile, the protein precursor is prepared which contains theNIa recognition sequence (cleavage sequence). This may be present in agiven place, or the protein may form part of a fusion protein with, forexample, glutathione-S-transferase or maltose-binding protein, linked byan appropriate linker (cleavage sequence). Reaction of the precursorwith NIa in a suitable buffer solution cleaves the precursor in vitro.If desired, N-terminal amino acid residues can be removed, as describedabove.

[0167] As before, the resulting protein can be isolated and purified byknown methods.

[0168] In respect of the second embodiment of the present invention, webelieve the naturally occurring reducing peptide has the sequence shownin Sequence ID 12, and consists of 526 amino acids, with a valineresidue as the N-terminal.

[0169] The naturally occurring DNA encoding the peptide has, we believe,the nucleotide sequence 70 to 1647 indicated in sequence ID number 11.

[0170] The peptide of the invention is capable of reducing oxidizedglutathione and dichloroindophenol. This is an accurate description ofthe peptide, but is somewhat cumbersome, so that the peptide of theinvention will also be referred to herein as the KM31-7 peptide orprotein.

[0171] The KM31-7 protein originates in the body, or is a mutant orvariant thereof, so that there are minimal problems with toxicity and/orantigenicity.

[0172] In the second embodiment of the present invention, thepolypeptide having the sequence −23 to 526 of sequence ID 12 is believedto be a precursor of the KM31-7 protein. As such, the present inventionalso encompasses this precursor, as well as mutants and variantsthereof, and polynucleotide sequences encoding any of these.

[0173] The following discussion is essentially with regard to the secondembodiment of the present invention. It will be understood that many ofthe procedures described above in relation to CYVV NIa are alsoappropriate to the isolation, cloning and. expression of DNA encodingthe peptide of the invention. Any differences essentially arise from thefact that CYVV NIa is a plant virus protein, while the peptide of theinvention is a mammalian protein. General steps for obtaining anexpressed specific polypeptide from mammalian cells by geneticrecombination techniques will now be described.

[0174] mRNA encoding the KM31-7 peptide can be obtained andreverse-transcribed into ds-DNA by well known methods. Any appropriatemammalian cells, cell lines or tissue can be used as the source of theoriginal mRNA, but we prefer to use the cell line KM-102 derived fromhuman bone marrow stromal cells [Harigaya, K. and Handa, H. (1985),Proc. Natl. Acad. Sci. USA, 82, 3447-3480].

[0175] To extract mRNA from mammalian cells, various methods can beused, such as the guanidine thiocyanate hot phenol method or guanidinethiocyanate guanidine hydrochloric acid method, but the guanidinethiocyanate cesium chloride method is generally preferable.

[0176] Since the majority of mRNA present in the cytoplasm of eukaryoticcells is known to have a 3′ terminal poly A sequence, purification ofmammalian mRNA can be effected by adsorption onto an oligo(dT) cellulosecolumn, thereby taking advantage of this characteristic. The eluted mRNAcan then be further fractionated by methods such as sucrose densitygradient centrifugation.

[0177] Confirmation that the mRNA does indeed encode the desired peptidecan be achieved by translating the mRNA in a suitable system, such asthe Xenopus laevis oocyte system, the rabbit reticulocyte system or thewheat germ system (supra).

[0178] Measurement of the reducing activity of the expression productcan be performed as described below.

[0179] i) Determination of Dichloroindophenol Reducing Activity

[0180] The methodology for this determination is described by Beinert,H. in “Methods in Enzymology” (1962), 5, 546.

[0181] A 50 μM dichloroindophenol (Sigma) preparation is made up with 20mM phosphate buffer and 0.5 M NaCl (pH 7.8). 1 ml of this preparation isthen placed in a cuvette (SARSTEDT, 10×4×45 mm) and the sample is thenadded for measurement. 15 μl of 1 mM NADPH (Boeringer-Mannheim), made upin the same buffer, is added to the cuvette at room temperature to startthe reaction. Reductase activity can then be determined by following thedecrease in absorption of oxidized dichloroindophenol at 600 nm or byfollowing the decrease in absorption of NADPH at 340 nm.

[0182] ii) Determination of Oxidized Glutathione Reducing Activity

[0183] The methodology for this assay is described by Nakajima, T. etal. in “New Basic Experimental Methods in Biochemistry (6)—Assay MethodsUsing Biological Activity”, 3-34.

[0184] A preparation of 10 mM oxidized glutathione (Boeringer-Mannheim)is made up with 20 mM phosphate buffer and 0.5 M NaCl to a pH of 7.8. 15μl of this preparation are placed in a cuvette (10×4×4 mm) after thesample has first been placed in the cuvette. 15 μl of 1 mM NADPHprepared in the same buffer is then added to the cuvette at roomtemperature to start the reaction. Glutathione reductase activity canthen be determined by following the decrease in absorption at 340 nm.

[0185] Various methods, as described above, can be used to derive ds-DNAfrom mRNA. These include the S1 nuclease method, the Land method and theO. Joon Yoo method (supra), but we prefer to use the Okayama-Berg method[Okayama H. and Berg, P. (1982), Mol. Cell. Biol. 2, 161-170] in thisinstance.

[0186] The thus obtained ds-cDNA can then be incorporated into a cloningvector and the resulting recombinant plasmid can be introduced intoEscherichia coli as described above.

[0187] A strain containing the desired DNA encoding the peptide of theinvention can be selected by various methods, such as those describedabove in relation to selecting transformants containing NIa DNA, withany appropriate modifications of procedure. For example, if PCR is usedto prepare a probe, then suitable template DNA may either be the cDNAdescribed above, or genomic DNA.

[0188] In the second embodiment of the present invention, it is possibleto use a primary screen to reduce the number of transformant strains tobe tested. The primary screen is possible, because the peptide of theinvention is related to the cytokines, so that its mRNA shares the AUUUAmotif common to the mRNA of cytokines [Shaw, G. and Kamen, R. (1986),Cell 46, 659-667.] Thus, a synthetic oligonucleotide probe which iscomplementary to the AUUUA motif can be used in the primary screen.Screening by assay of the production of exogenous protein in mammaliancells can then be performed.

[0189] DNA encoding the peptide can then be excised from the vector andsequenced in similar fashion to that described above in relation to NIaDNA. Again, as described above, the DNA fragment can then be introducedinto an appropriate vector and used to transform a prokaryotic oreukaryotic host, as desired.

[0190] The KM31-7 protein may be used either alone or in combinationwith one or more other therapeutic drugs in the prevention and treatmentof conditions caused by, or related to, oxidative stress, or any diseasecaused by activated oxygen. Such conditions include, but are not limitedto, arteriosclerosis, diabetes, ischemic disorders (such as reperfusiondisorders, ischemic heart disease, cerebroischemia and ischemicenteritis), edema, vascular hyperpermeability, inflammation, gastricmucosa disorders, acute pancreatitis, Crohn's disease, ulcerativecolitis, liver disorders, Paraquat's disease, pulmonary emphysema,chemocarcinogenesis, carcinogenic metastasis, adult respiratory distresssyndrome, disseminated intravascular coagulation (DIC), cataracts,premature retinopathy, auto-immune diseases, porphyremia, hemolyticdiseases, Mediterranean anemia, Parkinson's disease, Alzheimer'sdisease, epilepsy, ultraviolet radiation disorders, radioactivedisorders, frostbite and burns.

[0191] Pharmaceutical compositions of the second embodiment of thepresent invention comprise a pharmaceutically active amount of theKM31-7 peptide and a pharmaceutically acceptable carrier therefor.

[0192] The compositions of the present invention may be administered invarious forms, such as by oral administration in the form of tablets,capsules, granules, powders and syrups, or by parenteral administrationin the form of injections, infusions and suppositories. Other suitableadministration forms will be apparent to those skilled in the art.

[0193] In the event that the peptide of the invention is to beadministered as an injection or infusion, then a pyrogen-freepreparation of the peptide is made up in a pharmaceutically acceptableaqueous solution suitable for parenteral administration. The preparationof the polypeptide solution so as to conform with the requirements ofpH, isotonicity and stability is within the technical expertise of thoseskilled in the art.

[0194] Dosage and form of administration can readily be determined byone skilled in the art, taking into account such criteria as patientcondition, body weight, sex, age, diet, severity of other infections,administration time and other clinically significant factors. Ingeneral, the normal adult oral dose will be in the range of about 0.01mg to about 1000 mg per day. This amount can be administered in a singledose or as several sub-doses over a period of 24 hours. When the peptideis administered parenterally, about 0.01 mg to about 100 mg peradministration can be given by subcutaneous, intramuscular orintravenous injection.

[0195] In order to fully characterize the KM31-7 protein, it wasimportant to obtain an antibody that was specific for this protein. Suchan antibody would be useful in assaying the function, quantification,purification and tissue distribution of the KM31-7 protein.

[0196] Accordingly, a hybridoma producing anti-KM31-7 antibody wasobtained by inoculating laboratory animals with the polypeptide producedby E. coli transformed by pMAL31-7, preparing a hybridoma ofantibody-producing cells together with myeloma cells, followed byscreening and cloning the hybridoma. The antibodies produced by theresulting hybridoma were capable of recognizing the polypeptide obtainedfrom serum-free culture supernatant of COS-1 cells transformed withpSRα31-7.

[0197] Thus, the prevent invention further provides an antibody,preferably a monoclonal antibody, or an equivalent thereof, whichspecifically recognizes KM31-7 protein, or a mutant or variant of KM31-7protein.

[0198] The antibody of the present invention is directed against theKM31-7 protein or a mutant or variant thereof. It will be appreciatedthat the antibody may be polyclonal or monoclonal, but that themonoclonal form is preferred. This is because of the uncertaintygenerally associated with polyclonal antibodies. For consistency ofresults, whether for therapy or for purification of KM31-7 protein, forexample, it is preferred to use monoclonal antibodies.

[0199] The antibodies of the invention may be prepared from any suitableanimal. However, problems with antigenicity can arise where the antibodyis non-human. In this respect, it is possible to engineer the antibodiesto more closely resemble human antibodies. This may be achieved eitherby chemical or genetic modification by methods well known in the art.

[0200] The present invention also envisages anti-idiotypic antibodies,that is, antibodies whose recognition site recognizes the recognitionsite of the above antibodies. Such anti-idiotypic antibodies can beprepared by administering the original antibody to a suitable animal. Itwill be appreciated that this process can continue, effectively adinfinitum, with each generation corresponding to either the original orthe anti-idiotype. However, if the antibodies are not selected for thecorrect recognition after each generation, then specificity mayeffectively be lost. Anti-idiotypic antibodies may be useful, forexample, in assaying free anti-KM31-7 antibody.

[0201] The present invention also envisages fragments of the antibodiesof the invention which are capable of recognizing KM31-7 protein, andmolecules carrying the recognition site of such antibodies. Suchfragments and molecules are referred to herein as “equivalents” of theantibodies of the invention.

[0202] The plasmid pMAL31-7 was used to transform E. coli, and theexpression product was purified and used to immunize laboratory animals.Spleen cells from the immunized animals were used to prepare a hybridomaby fusion with myeloma cells, and a clone producing anti-M31-7monoclonal antibodies was obtained in high concentration and with goodstability (this clone was named MKM150-2 and deposited at theFermentation Research Institute of the Agency of Industrial Science andTechnology, Japan, under the deposit number FERM BP-5086). A culture ofthis clone yields anti-KM31-7 monoclonal antibodies from the culturesupernatant.

[0203] The resulting anti-KM31-7 monoclonal antibody reactsimmunochemically with the fusion protein obtained by introducing andexpressing pMAL31-7 in E. coli. This antibody also reactsimmunochemically with KM31-7 protein obtained from the culturesupernatants of mammalian cells transformed with cDNA encoding KM31-7.

[0204] In order to produce a monoclonal antibody, the proceduresoutlined below will generally have to be followed. These consist of:

[0205] (a) purification of the biopolymer to be used as the antigen;

[0206] (b) immunization of mice by injection of the antigen, andpreparing antibody-producing cells at the appropriate time from thespleen by sampling and assaying blood;

[0207] (c) preparation of myeloma cells;

[0208] (d) fusing spleen and myeloma cells;

[0209] (e) screening to select the hybridoma group that produces thedesired antibodies;

[0210] (f) preparing a single clone (cloning);

[0211] (g) culturing the hybridoma for large-scale production ofmonoclonal antibody, or husbanding mice infected with the hybridoma, asthe case may be; and

[0212] (h) assaying the physiological activity or properties as alabelling reagent of the resulting monoclonal antibody.

[0213] The production of anti-KM31-7 monoclonal antibodies will now bedescribed with reference to the procedures outlined above. It will beappreciated that it is not necessary to follow precisely the followingdescription, and that it is possible to use any suitable procedure. Forexample, it is possible to use antibody-producing cells and myelomasother than spleen cells as well as antibody-producing cells and myelomasof other mammals. The following procedure represents the currentlypreferred method for obtaining anti-KM31-7 monoclonal antibodies.

[0214] (a) Antigen Purification

[0215] The fusion protein obtained by expressing pMAL31-7 in E. coli andpurifying the product is an effective antigen. A culture of E. coli TB-1transformed with pMAL31-7 was induced with isopropylβ-D-thiogalacto-pyranoside (IPTG). The expressed fusion protein was thenpurified by affinity chromatography using an amylose resin column (NewEngland BioLabs). KM31-7 protein purified from the serum-free culturesupernatant of COS-1 cells transformed with pSRα31-7 is also aneffective antigen.

[0216] (b) Preparation of Antibody-Producing Cells

[0217] The purified fusion protein obtained in (a) is mixed withFreund's complete or incomplete adjuvant, or an adjuvant such as potashalum, and laboratory animals are then immunized with the resultingvaccine. BALB/c mice are a preferred choice for use as the laboratoryanimals, because the majority of useful myelomas derived from mice arederived from BALB/c mice. Moreover, the characteristics of these micehave been studied in a great amount of detail. Furthermore, if both theantibody-producing cells and the myeloma are from BALB/c mice, then theresulting hybridoma can be grown in the abdominal cavity of BALB/c mice.Thus, the use of BALB/c mice offers the advantage of enabling monoclonalantibodies to be easily obtained from ascites without having to employcomplex procedures. Nevertheless, the present invention is not limitedto the use of BALB/c mice.

[0218] The antigen may be administered in any suitable form, such as bysubcutaneous injection, intraperitoneal injection, intravenousinjection, intracutaneous injection or intramuscular injection, and weprefer subcutaneous injection or intraperitoneal injection.

[0219] Immunization may be performed once or on a plurality of occasionswith suitable intervals. The preferred regimen is to immunize and thenboost, one or more times, at intervals of from 1 to 5 weeks. Theeffectiveness of later procedures can be improved if the antibody titerto said antigen in the serum of the immunized animals is regularlyassayed, and animals having a sufficiently high antibody titer are usedto provide antibody-producing cells. Antibody-producing cells forsubsequent fusion are preferably isolated from an animal 3 to 5 daysafter the final immunization.

[0220] Methods for assaying antibody titer include, for example, variousknown techniques, such as radioisotope immunoassay (RIA), enzyme-linkedimmunosorbent assay (ELISA), the fluorescent antibody technique andpassive hemagglutination, but RIA and ELISA are preferable for theirsensitivity, speed, accuracy and potential for automation.

[0221] A suitable form of ELISA is as follows. Antigen is adsorbed ontoa solid phase and then the solid phase surface is exposed to a proteinunrelated to the antigen, such as bovine serum albumin (BSA), to blockany areas of the surface which have no adsorbed antigen. The solid phaseis then washed, after which it is exposed to a serially diluted sampleof the primary antibody (e.g. mouse serum). Any anti-KM31-7 antibody inthe sample binds to the antigen. After washing, secondary,enzyme-linked, anti-mouse-antibody is added and is allowed to bind tobound mouse antibody. After washing, enzyme substrate is added and theantibody titer can then be calculated by measuring a parameter, such ascolor change, caused by decomposition of the substrate.

[0222] (c) Myeloma Cell Preparation

[0223] Established mouse cell lines are preferably used as the source ofmyeloma cells. Suitable examples of such cell lines include myeloma cellline P3-X63 Ag8-U1 (P3-U1) from 8-azaguanine resistant mice of BALB/corigin [Current Topics in Microbiology and Immunology, 81, 1-7 (1978)],P3-NSI/1-Ag4.1 (NS-1) [European J. Immunology, 6, 511-519 (1976)],SP2/O-Ag14 (SP-2) [Nature, 276, 269-270 (1978)], P3-X63-Ag8.653 (653)[J. Immunology, 123, 1548-1550 (1979)] and P3-X63-Ag8 (X63) [Nature 256,495-497 (1975)]. These cell lines may be subcultured in suitable media,such as 8-azaguanine medium (RPMI-1640 medium containing 8-azaguanine,1.5 mM glutamine, 5×10⁻⁵ M 2-mercaptoethanol, 10 μg/ml gentamycin and10% fetal calf serum), Iscove's Modified Dulbecco's Medium (IMDM) orDulbecco's Modified Eagle's Medium (DMEM). The cell count is elevated toat least 2×10⁷, on the day of fusion, by subculturing with normalmedium, also known as complete GIT [5.5 ml of MEM non-essential aminoacids solution (NEAA, Gibco), 27.5 ml of NCTC109 (Gibco), 6 ml ofpenicillin-streptomycin solution (Sigma) and 11 ml of glutamine 200 mMsolution (Sigma) in 500 ml of GIT medium (Wako Pure Chemical Industry)],3 to 4 days before cell fusion.

[0224] (d) Cell Fusion

[0225] The antibody-producing cells are plasma cells and their precursorlymphocytes. These may be obtained from any appropriate site of anindividual animal, such as the spleen, lymph nodes, peripheral blood orany suitable combination of these. However, spleen cells are mostcommonly used.

[0226] Antibody-producing cells are harvested, 3 to 5 days after thefinal immunization, from mice having at least the prescribed antibodytiter. The resulting antibody-producing cells are then fused with themyeloma cells obtained in (c) above. The process most commonly used atpresent is to fuse the spleen cells with the myeloma cells usingpolyethylene glycol, owing to the relatively low level of cellulartoxicity and ease of manipulation of this compound. This process isperformed as follows.

[0227] Spleen cells and myeloma cells are thoroughly washed with mediumor phosphate buffered saline (PBS), mixed so that the ratio of spleencells to myeloma cells becomes roughly between 5 and 10:1, and thensubjected to centrifugal separation. The supernatant is discarded andthe clump of cells is thoroughly broken up, and then a mixed solution ofpolyethylene glycol (PEG, molecular weight: 1000 to 4000) is added withstirring. After several minutes, the cells are subjected to centrifugalseparation. The supernatant is again discarded, and the settled cellsare suspended in a suitable amount of complete GIT containing 5 to 10ng/ml of mouse IL-6 and then transferred to the wells of a cultureplate. Once cell growth has been confirmed in each well, the medium isreplaced with HAT medium (complete GIT containing 5 to 10 ng/ml of mouseIL-6, 10⁻⁶ to 10⁻³ M hypoxanthine, 10⁻⁸ to 10⁻⁷M aminopterin and 10⁻⁶ to10⁻⁴ M thymidine).

[0228] (e) Selection of Hybridoma Groups

[0229] The culture plate is incubated in a CO₂ incubator at 35 to 40° C.for 10 to 14 days. During this time, fresh HAT medium equivalent to halfthe amount of medium is added every 1 to 3 days.

[0230] The myeloma cells are from an 8-azaguanine-resistant cell lineand both the myeloma cells and hybridomas consisting only of myelomacells cannot survive in HAT medium. However, hybridomas comprising anantibody-producing cell part, including hybridomas of antibody-producingcells and myeloma cells, can survive. Hybridomas consisting only ofantibody-producing cells are mortal, so that hybridomas consisting ofboth myeloma and antibody-producing cells can be selected by culturingin HAT medium.

[0231] HAT medium is replaced with HT medium (wherein aminopterin hasbeen omitted from HAT medium) in those wells in which hybridomas whichhave been observed to develop colonies. A portion of the culturesupernatant is then removed and anti-KM31-7 antibody titer is assayedby, for example, ELISA.

[0232] The above process is described with respect to an8-azaguanine-resistant cell line, but other cell lines can also be used,provided that they permit the selection of hybridomas. The compositionof the medium used naturally also changes in such cases.

[0233] (f) Cloning

[0234] Hybridomas from (e) which have been determined to produceanti-KM31-7 specific antibody are transferred to a different plate forcloning. Various cloning methods can be used, such as the seeding method(wherein the hybridomas are subjected to limiting dilution analysis sothat each well contains only one hybridoma), the soft agar method(wherein seeded colonies are taken in soft agar medium), the seedingmethod (wherein a single cell is removed by a micro-manipulator), andthe sorter cloning method (wherein individual cells are separated by acell sorter). Limiting dilution analysis is used most frequently due toits simplicity.

[0235] Cloning, such as by limiting dilution analysis, is repeated 2 to4 times for those wells in which an antibody titer continues to beobserved. A clone consistently exhibiting the production of anti-KM31-7antibody is selected as the hybridoma of choice.

[0236] (g) Preparation of Monoclonal Antibody by Hybridoma Culture

[0237] The hybridoma of choice from (f) is then cultured in ordinarymedium. Large-volume culture is performed by rotary culturing usingeither a large culture bottle or a spinner. Anti-KM31-7 monoclonalantibodies can be obtained by subjecting the cell supernatant to gelfiltration, and then collecting and purifying the IgG fraction. Inaddition, the hybridoma can also be grown in the abdominal cavity of thesame strain of mouse (e.g. the above-mentioned BALB/c mice) or Nu/Numice, for example. A simple method for performing this step is to use amonoclonal antibody preparation kit (e.g., MAbTrap GII of Pharmacia).

[0238] (h) Identification of Monoclonal Antibody

[0239] Determination of the isotype and subclass of the monoclonalantibody obtained in (g) can be performed as described below. Examplesof identification techniques which can be used include the Ouchterlonymethod, ELISA and RIA. Although the Ouchterlony method is simple, thereis a drawback, in that the monoclonal antibody must be concentrated incases in where concentration is excessively low.

[0240] If either ELISA or RIA is used, the culture supernatant can bedirectly exposed to a solid phase on which antigen has been adsorbed.Secondary antibodies specific for each type of IgG subclass can then beused to identify subclass type. In the alternative, an isotyping kit(for example, the mouse monoclonal antibody isotyping kit of Amersham)can be used. Protein quantification can be performed by the Folin-Lowrymethod or by measuring absorbance at 280 nm [1.4 (OD₂₈₀)=1 mg/ml ofimmunoglobulin].

[0241] In the accompanying Examples, the monoclonal antibody obtainedfrom the hybridoma designated MKM150-2 was determined to be of the IgGclass isotype and was identified as belonging to the IgGl subclass.

[0242] The monoclonal antibody obtained in accordance with the presentinvention has a high-specificity for KM31-7 protein. Moreover, sincemonoclonal antibody is obtained consistently and in good quantities byculturing the above-mentioned hybridoma, it can be used for theisolation and purification of KM31-7 protein, and the isolation andpurification of KM31-7 protein with said monoclonal antibody by immuneprecipitation using an antigen-antibody reaction forms a part of thepresent invention.

[0243] The present invention will now be illustrated with reference tothe accompanying, non-limiting Examples. All solutions are aqueous andprepared from deionized water, and those expressed in the form ofpercentages are w/v, unless otherwise specified. If methods are notspecified, then they can be found in “Molecular Cloning—A LaboratoryManual” [second edition, Sambrook, J., Fritsch, E. F. and Maniatis, T.(1989), Cold Spring Harbor Laboratory Press]. Methods of preparation ofsolutions and other media are given in the later section entitled“Media” where they are not given in the Examples. Methods referred to inthe Examples without techniques should be construed in the context ofpreceding Examples.

[0244] A) Source Material for CYVV

[0245] A leaf infected with the clover yellow vein virus and which hadbeen stored in the frozen state [CYVV-isolate No. 30: Uyeda, I. et al.(1975), Ann. Phytopath. Soc. Japan 41: 192-2031 was milled with 10volumes of inoculation buffer.

[0246] Propagation of CYVV was carried out using Vicia faba cultivarWase-Soramame which had developed a second adult leaf. An adult leaf wassprayed with carborundum (400 mesh) and was then inoculated with theliquid obtained above using a glass spatula. About eight to ten daysafter the inoculation, the inoculated leaf had developed a mesh-likemosaic condition. This leaf was removed and used as a source of CYVV.

[0247] B) Purification of Virus

[0248] Leaves isolated in A) above were chopped with a mincer containing3 volumes of extraction buffer. After chopping, the leaves were furtherground in a mortar and pestle. The resulting preparation was stirredslowly at room temperature for 1 hour using a motor equipped with astainless steel agitating blade. The liquid preparation was thensqueezed out through a double layer of gauze.

[0249] All of the subsequent procedures were carried out at 4° C.

[0250] The crude liquid was mixed with a half volume of chloroform andthe mixture was blended in a Waring blender, after which the preparationwas centrifuged at 6,000×g for 15 minutes. Following centrifugation, theaqueous layer was collected, and polyethylene glycol (PEG #6,000) wasadded to this fraction to a final concentration of 4% v/v.

[0251] The resulting mixture was stirred over ice for 1 hour and allowedto stand on ice for another 1 hour. The thus obtained liquid wassubjected to centrifugation at 6,000×g for 15 minutes, and theprecipitate was recovered as the virus-containing fraction. Thisprecipitate was suspended in 50 ml of a 10 mM phosphate buffer (pH 7.4)containing 0.5 M urea and then mixed with an equal volume of carbontetrachloride, and the resulting preparation was stirred vigorously for5 minutes, after which time the preparation was subjected tocentrifugation at 3,000×g for 10 minutes. The aqueous phase was thenultracentrifuged at 4° C. at 30,000 rpm for 90 minutes using an HitachiRP-30 rotor. The resulting pellet was recovered as the virus-containingfraction.

[0252] The pellet was suspended in 10 mM phosphate buffer containing 1%Triton X100 (pH 7.4) and this suspension was subjected to centrifugationat 4° C. at 8,000×g for 1 minute. The resulting supernatant was layeredon a graduated 10 to 40% sucrose density gradient column tube (40%: 10ml, 30%: 10 ml, 20%: 10 ml, 10%: 10 ml; and which had been allowed tostand overnight at 4° C.) which had previously been prepared with 10 nMphosphate buffer (pH 7.4). Once the tube had been layered with thesupernatant, it was subjected to ultracentrifugation at 4° C. at 23,000rpm for 120 minutes in a Hitachi PRS25 rotor.

[0253] After centrifugation, the sucrose density gradient column tubewas fractionated using a fractionater (Model UA-2: manufactured by ISCOCo.) equipped with an OD_(260 nm) detector. The fractions having asucrose density of about 20 to 30% and absorbing at OD_(260 nm) weretaken as the virus-containing fraction.

[0254] The recovered virus fraction was diluted 2-fold with 10 mMphosphate buffer (pH 7.4) and subjected to centrifugation at 4° C. at40,000 rpm for 90 minutes using a Hitachi RP-65 rotor. The resultingprecipitate was resuspended in 10 mM phosphate buffer (pH 7.4) to yielda purified virus solution.

[0255] C) Isolation of Viral RNA

[0256] The genomic RNA of CYVV comprises about 10,000 bases and ispolyadenylated at its 3′-terminus. The combination of length andpolyadenylation make it difficult to extract the full-length RNA withoutany loss when using either the conventional phenol/sodium dodecylsulfate method or the guanidinium thiocyanate method. Accordingly, theviral genomic RNA was prepared by the alkaline sucrose density gradientcentrifugation method.

[0257] 500 μl of the virus solution (which we had calculated to contain2 mg of the virus on the basis that 1 mg/ml of virus has an OD_(260nm)of 2.5) was added to 500 μl of degradation solution and the mixture wasallowed to stand at room temperature for 20 minutes. After this time,the mixture was layered over a 0% to 33.4% sucrose density gradientcolumn tube (33.4%: 1.4 ml, 30.4%: 7.6 ml, 27%: 7.0 ml, 23%: 6.3 ml,18.7%: 5 ml, 12%: 3.2 ml, 0%: 2.7 ml; and which had been allowed tostand at 4° C. overnight) which had been prepared with 1×SSC. The bufferused was also 1×SSC, and the layered tube was then subjected toultracentrifugation in a Hitachi RPS-27 rotor at 15° C. at 24,000 rpmfor 9 hours. After this time, the bottom of the tube was punctured inorder to fractionate the sucrose density gradient. The viral genomic RNAfraction was identified by measuring the absorbance of each fraction at260 nm. Sedimentation of the viral genomic RNA occurred at a sucroseconcentration of about 20 to 30%. Genomic RNA was obtained in a yield ofabout 25 μg.

[0258] D) Synthesis of Viral cDNA

[0259] cDNA was synthesized using the genomic RNA prepared in C) aboveas a template. cDNA synthesis was carried out using the cDNA SynthesisSystem Plus (manufactured by Amersham). The resulting cDNA was purifiedon a Sephadex G50 column (registered trademark, manufactured byPharmacia), and a poly C chain was added to the 5′ terminus of thepurified cDNA using dCTP and terminal deoxynucleotide transferase(manufactured by Bethesda Research Laboratories). A preparation ofplasmid pBR322 (manufactured by Bethesda Research Laboratories) was madeby digesting the plasmid with the restriction enzyme PstI and thenadding a 3′ poly G chain at both termini. The cDNA was then added tothis preparation, and the mixture was incubated at 65° C. for 5 minutes,after which time it was incubated at 57° C. for 2 hours. The resultingmixture was then gradually cooled to allow annealing of the poly C chainof the cDNA with the poly G chain of the plasmid.

[0260] E) Transformation

[0261]Escherichia coli strain HB 101 was transformed with the novelplasmid prepared in D) above by the calcium chloride method. A seedculture of E. coli strain HB 101 was prepared by shaking overnight inliquid LB medium. 0.5 ml of this seed culture was used to inoculate 50ml of fresh liquid LB medium and the inoculum was cultured with shakingat 37° C. until an OD_(550 nm) of 0.5 was obtained. Bacterial cells wererecovered by centrifugation at 4° C. at 5,000×g for 5 minutes and theresulting pellet was gently suspended in 25 ml of Tris-calcium bufferand allowed to stand on ice for 5 minutes. The resulting suspension wascentrifuged again at 4,000×g for 4 minutes and the pellet was suspendedin 5 ml of Tris-calcium buffer and allowed to stand on ice for 2 hoursto yield competent cells.

[0262] 100 μl of the novel plasmid obtained in D) above were added to200 μl of the competent cells obtained above, and the mixture wasallowed to stand on ice for 30 minutes. After this time, the mixture wasincubated at 42° C. for 2 minutes and then 1 ml of liquid LB medium wasadded, and the resulting mixture was cultured with shaking at 37° C. fora further one hour. The resulting mixture was spread onto solid LBmedium containing 12.5 μg/ml tetracycline hydrochloride and 1.5% w/wagar. The cells were cultured at 37° C. overnight to provide a cDNAclone library.

[0263] F) Preparation and Selection of Plasmid pNS51

[0264] The cDNA recombinant plasmid library was analyzed using thealkaline-SDS method. In more detail, the method was as follows.

[0265] 2 ml of liquid LB medium was inoculated with a colony of bacteriaresistant to tetracycline but sensitive to ampicillin, obtained from thesolid LB culture of E) above, and the resulting suspension was culturedwith shaking at 37° C. overnight. Cells were recovered by centrifugationat 10,000×g and suspended in 70 μl of lysis buffer containing a further16 μl of lysozyme solution (10 mg/ml). After agitating for 5 seconds tocreate a suspension, the suspension was allowed to stand at roomtemperature for 5 minutes. After this time, 160 μl of alkaline-SDSsolution were added to the suspension and the resulting mixture wasfirst mixed by inverting the tube several times and then allowed tostand on ice for 5 minutes. 120 μl of 5 M potassium acetate were nextadded to the mixture which was then again allowed to stand on ice for 5minutes. After this time, the mixture was centrifuged at 10,000×g for 5minutes at 4° C.

[0266] After centrifugation, the supernatant was transferred to a freshtube, and 250 μl of isopropanol was further added to the tube and theresulting mixture was allowed to stand on ice for 30 minutes. After thistime, the mixture was again centrifuged at 10,000×g for 5 minutes, andthe resulting pellet was dissolved in 100 μl of TE buffer [10 mM Tris, 1mM EDTA (pH 8.0)]. Phenol extraction was then performed by adding anequal amount of phenol/chloroform/isoamyl alcohol (25:24:1) to theresulting mixture with vigorous mixing, and centrifuging the mixture at10,000×g for 5 minutes at 4° C. (hereinafter, this procedure is referredto as phenol extraction).

[0267] 100 μl of the aqueous layer obtained from the centrifugation wasmixed with 10 μl of 3 M sodium acetate (pH 5.2) and 250 μl of ethanol,and the resulting mixture was allowed to stand on dry ice for 10minutes, followed by centrifugation at 10,000×g for 5 minutes to recoverthe nucleic acid fraction (hereinafter, this procedure, using the samerelative volumes of supernatant, ethanol and sodium acetate solution, isreferred to as ethanol precipitation).

[0268] The resulting pellet was suspended in 50 μl of a solution ofRNase A (10 μg/ml in TE buffer) and then incubated at 37° C. for 1 hour.After this time, 30 μl of a solution of polyethylene glycol (2.5 Msodium chloride, 20% polyethylene glycol #8,000) was added to theincubated suspension, and the mixture was allowed to stand on ice for 1hour. After this time, the mixture was centrifuged at 10,000×g for 5minutes at 4° C. to recover the DNA fraction as a pellet, and ethanolprecipitation was performed twice more to yield purified plasmid DNA.

[0269] The purified plasmid DNA was cleaved with restriction enzyme Pstland subjected to 1 agarose gel electrophoresis using TBE solution. Afterelectrophoresis, the gel was subjected to Southern blot hybridization(Southern, E. M. (1975), J. Mol. Biol. 89: 503-517]. More specifically,the gel was shaken in denaturation solution for 40 minutes, thentransferred to and shaken in neutralization buffer for 2 hours.

[0270] After shaking, the gel was transferred onto a polyurethane estersponge containing 20×SSC to transfer the DNA to a piece of Hybond-Nmembrane (registered trademark of Amersham) situated on the sponge.After the DNA had been allowed to transfer to the Hybond-N membrane, themembrane was shaken in 1×SSC for 10 minutes and then dried at 80° C. for1 hour to fix the DNA. The membrane was then placed in prehybridizationsolution [5 ml of formamide, 1 ml of 50× Denhardt's solution, 2.5 ml of20×SSC, 100 μl of yeast tRNA (50 mg/ml), 100 μl of 10% SDS and 1.3 ml ofredistilled water] and incubated at 50° C. for 3 hours.

[0271] Viral genomic RNA which had been cleaved with Mg²⁺ was used as aprobe. More particularly, 4 μl of 5 μl denaturation buffer was added to16 μl of the solution obtained in C) above containing 1 μg viral RNA,and the mixture was incubated at 37° C. for 3 hours. 5 μl of denaturedRNA solution containing 100 ng of RNA (calculated on the basis that 1mg/ml of RNA has an OD₂₆₀ of 20) was recovered by ethanol precipitationfollowed by heating at 70° C. for 5 minutes and then quenching on ice.

[0272] 4 μl of 5× labelling buffer, 1 μl of 3.3 mM [γ-³²P] ATP (0.37MBq), and 10 μl of redistilled water were added to the denatured RNAsolution. 20 U of T4 polynucleotide kinase solution [manufactured byTakara Shuzo] were then added to the mixture, followed by incubation at37° C. for 30 minutes. Unincorporated [γ-³²P] ATP was removed byrepeating ethanol precipitation five times.

[0273] The resulting labelled probe was added to hybridization solution[5 ml of formamide, 2 ml of 50% dextran sulfate, 200 μl of 50×Denhardt's solution, 2.5 ml of 20×SSC, 50 μl of yeast tRNA (50 mg/ml),100 μl of 10% SDS, 1.3 ml of redistilled water] to a final concentrationof 5×10⁵ cpm/ml, and the Hybond-N membrane obtained above wastransferred to this solution and subjected to hybridization byincubation at 50° C. overnight, followed by washing with shaking at 6°C. with 2×SSC containing 0.1% sodium dodecyl sulfate (SDS). Thisprocedure was repeated three times, and the Hybond-N membrane was thenfurther washed with 0.1×SSC containing 0.1% SDS with shaking at roomtemperature for 1 hour, and then dried.

[0274] Autoradiography revealed a plasmid which we designated pNS51which had an insertion fragment comprising about 6,500 base pairs (bp)which had hybridized with the viral genomic RNA.

[0275] G) Sequence Determination of pNS51 Insert

[0276] In order to prepare a restriction map of the cDNA cloned inpNS51, the plasmid was digested with each of the restriction enzymes;BamHI, EcoRI, Hind III, KpnI, NcoI, PstI, SalI, SphI, SacI, SmaI, XhoI,XbaI, and pairs thereof. The resulting map is shown in FIG. 1.

[0277] Subsequently, each of the fragments obtained by digestion withthe restriction enzymes SalI and PstI was inserted into M13mp19 RF DNA.This was done by digesting pNS51 with the restriction enzymes PstI andSalI and running the cleavage products on a 5% polyacrylamideelectrophoresis gel using 1× TBE. After electrophoresis, the gel wasstained with 1 μg/ml of ethidium bromide in order to be able to identifythe bands under UV light. Strips of the gel containing each DNA bandwere excised with a razor and subjected to electroelution with anelectroeluter (manufactured by Amicon) equipped with Centricon-30(manufactured by Amicon).

[0278] Each fragment was then inserted, using a DNA ligation kit[manufactured by Takara Shuzo], into M13mp19 which had previously beendigested either with SalI only, or with both of PstI and SalI, and whichhad then been treated with alkaline phosphatase.

[0279] The resulting recombinant M13mp19 RF-DNA (wherein the fragmentshad been inserted using T4 DNA ligase) was introduced into E. colistrain JM109 using the rubidium chloride method. A single colony ofstrain JM109 which had been cultured on M9 minimum agar medium wasinoculated into and cultured in liquid SOB medium with shakingovernight. 0.6 ml of this overnight culture was inoculated into 50 ml offresh liquid SOB medium and cultured with shaking at 37° C. until theOD_(600 nm) reached 0.5. The cells were then recovered by centrifugationat 5,000×g at 4° C. for 10 minutes and the pellet was gently suspendedin 25 ml of TFB1 buffer and allowed to stand on ice for 20 minutes.

[0280] The suspension was again centrifuged at 3,000×g for 5 minutes,and the pellet was suspended in 2 ml of TFB2 buffer and then allowed tostand on ice for 20 minutes to provide competent cells.

[0281] The recombinant M13mp19 RF-DNA obtained above was used in amountsof between 1 and 10 μl after ligation and mixed with 100 μl of thecompetent cells and allowed to stand on ice for 1 hour. After this time,the mixture was incubated at 42° C. for 1.5 minutes and then 500 μl ofliquid SOC medium was added to the mixture which was cultured withshaking at 37° C. for a further 1 hour.

[0282] 2× YT medium containing 0.8% agar, 30 μl of 100 mM IPTG, 10 μl of10% 5-bromo-4-chloro-3-indolyl-↓-galactoside (X-gal) and 100 μl of anindicator bacteria (an overnight culture of strain JM109 cultured withshaking in 2× YT medium at 37° C.) were added to the resulting culturewith shaking, and the mixture was poured onto solid 2× YT mediumcontaining 1.2% agar. After the mixture had solidified, it was culturedat 37° C. overnight. Recombinant phages subsequently showed as whiteplaques.

[0283] Each of the resulting white plaques was inoculated into liquid 2×YT medium containing the indicator bacteria in an amount of 1/100, andthis was cultured with shaking at 37° C. overnight. After centrifugationat 10,000×g for 1 minute, the supernatant was frozen or stored at 4° C.as a phage solution, and the cell pellet was processed in a standardprocedure for preparing a plasmid to recover RF double-stranded DNA(hereinafter, abbreviated as RF-DNA), which is a replicationintermediate of the phage. The presence of an insertion fragment wasconfirmed by digestion with a restriction enzyme.

[0284] Thus, 51SS/M13mp19 (i.e. M13mp19 containing a SalI/SalI fragmentwhich, in turn, is a portion of 51PS5′/M13mp19 genome containing aPstI-SalI fragment from the 5′ region of the CYVV genome) was obtained.RF-DNA's were purified from these phage clones by alkaline-SDS andcesium chloride density gradient centrifugation in accordance withstandard procedures.

[0285] 5 μg of the resulting purified DNA was first cleaved with BamHIto obtain a 5′ sticky end, and then cleaved further with KpnI to obtaina 3′ sticky end. Next, in accordance with the protocol accompanying theErase-a-base system (manufactured by Promega), exonuclease was added forthe stepwise deletion of bases from the 5′ sticky end of each cDNAlinearized plasmid. Treatment with exonuclease was continued for 10minutes, and samples were removed at different times between 1 and 10minutes. Since the vector has a 3′ sticky end, it is not attacked by theexonuclease. The various samples obtained over the period of 1 to 10minutes were then blunt-ended by treatment with S1 nuclease and ligatedinto a circular form using T4 DNA ligase.

[0286] The resulting, circular, recombinant M13mp19 RF-DNA wasintroduced into E. coli strain JM 109 using the rubidium chloride methoddescribed above. Accordingly, recombinant phages having variable lengthcDNA inserts were obtained as a white plaques.

[0287] RF-DNA was extracted from the recombinant phage subclonesobtained above by conventional means to select clones having inserts ofdifferent length. The selected clones were separately cultured byinoculation into liquid 2× YT medium containing an indicator strain ofE. coli and were cultured with shaking at 37° C. overnight. After thistime, 1.5 ml of each culture was centrifuged at 4° C. at 10,000×g for 5minutes. One ml of the supernatant was transferred to a fresh tube and250 μl of a 20% polyethylene glycol aqueous solution (20% polyethyleneglycol #8000, 2.5 M sodium chloride) was then added to the tube withmixing and then allowed to stand on ice for 30 minutes. The mixture wassubsequently centrifuged at 10,000×g for 5 minutes at 4° C., and theresulting pellet was suspended in 100 μl of TE buffer. Single strandedDNA (hereinafter abbreviated as ssDNA) phage genomes were then recoveredfrom each preparation by phenol extraction and ethanol precipitation asbefore.

[0288] Nucleotide sequencing of each of the subclones obtained above wasthen performed by the dideoxy chain termination procedure, using thessDNA as a template. More specifically, the phage ssDNA was labeled with[α-³²P]dCTP (220 TBq/mmol, manufactured by Amersham) using the7-deaza-sequenase version 2.0 dCTP kit (manufactured by USB). Theresulting reaction product was run on a 5% polyacrylamideelectrophoresis gel containing 8 M urea and using 1× TBE as the buffer.The gel was dried, and the nucleotide sequence was then determined byautoradiography.

[0289] By analyzing the nucleotide sequences of the 5′-PstI-SalIfragment and the M-SalI-SalI fragment of pNS51, we were able todetermine a total of 3,839 bases.

[0290] As a result of the search for an open reading frame (hereinafterabbreviated as ORF) in the sequence obtained above, a single ORF for apolypeptide was detected on the + sense strand of the viral genome. Thepolypeptide sequence encoded in the ORF was then determined. A homologysearch comparing this polypeptide sequence with known sequences usingGeneBank revealed homology with TEV-HAT.

[0291] It is known that viruses such as TEV, plum pox virus orpoliovirus (an animal virus) mature through the action of a protease,and that the protease generally cleaves a peptide bond containing aGln-Ala, Gln-Ser or Gln-Gly link [Willink J. and van Kammen, A. (1988),Arch. Virol. 98: 1-26]. We were also able to detect three cleavagesequences in the deduced polypeptide sequence, as a result of analysisof the cleavage site(s) of the coat protein of CYVV-No. 30 [Uyeda, I. etal. (1991), Intervirology 32: 234-245]. From one of these cleavagesites, a polypeptide of from Gly at position 4 to Gln at position 437 ofthe amino acid sequence was determined to be Nuclear Inclusion a fromCYVV.

[0292] H) Preparation of a CYVV-NIa/IL-11 Fusion Protein

[0293] The DNA identified in G) above as coding for CYVV NIa wastandemly linked, in-frame, at its 3′ end to DNA encoding humaninterleukin 11 (IL-11), via a linking sequence encoding Gln-Ala.

[0294] The following were examined:

[0295] first, whether or not the DNA identified in G) above encodes anNIa having proteolytic activity;

[0296] second, whether or not the protease cuts the desired amino acidsequence;

[0297] third, whether or not the protease activity of the NIa is stillexhibited when the NIa is part of a fusion protein together with adesired heterologous protein in a manner similar to that existing in avirus-infected cell; and

[0298] fourth, whether or not any heterologous protein obtained throughthe cleavage of such a fusion protein maintains integrity, structure andfunction.

[0299] Since the NIa itself is produced by excision from a viralprecursor polypeptide, the NIa cistron has no initiation codon.Therefore, it is necessary to add an initiation codon, ATG, to the 5′terminus of the NIa gene. The 3′ terminus of the NIa gene must also beconnected with the 5′ terminus of the IL-11 gene within the same ORF. Inorder to solve these problems, the NIa was modified using the polymerasechain reaction (PCR) technique by utilizing a XhoI cleavage site presentin the center of the NIa gene.

[0300] In order to add the initiation codon ATG and a recognition sitefor the restriction enzyme NcoI suitable for cloning to the 5′ terminusof NIa (NIa5′), PCR primers were prepared. The sequences prepared were5′GTCCATGGGGAAAAGTAAGAGAACA3′ (referred to as NSATG; sequence ID number:3) and 5′ACTCTGAGACCGTGCTCGAG3′ (referred to as NSX1; sequence IDnumber: 4).

[0301] 0.8 μl of a dNTP solution (25 mM each of DATP, dTTP, dCTP anddGTP), 10× Taq buffer (manufactured by Promega) and 1 μg of each of theresulting primers were added to 1 μg of plasmid pNS51 DNA, and themixture was made up to 100 μl with redistilled water. 5 U of DNApolymerase made up in 10× Taq buffer were added to effect PCR. For thePCR program, a cycle of 92° C. for 1 minute, 37° C. for 1 minute and 72°C. for 2 minutes was repeated 20 times followed by a single cycle of 92°C. for 1 minute, 37° C. for 1 minute and 72° C. for 30 minutes.

[0302] The resulting amplified DNA was subjected to phenol extractionand ethanol precipitation, and then run on a 5% polyacrylamideelectrophoresis gel. One band containing DNA was detected by ethidiumbromide staining, and this band was excised from the gel and subjectedto electroelution with a Centreluter (manufactured by Amicon) equippedwith Centricon-30 (manufactured by Amicon). After the elution, theeluted DNA fragment was concentrated and recovered by centrifugation at7,500×g using Centricon (Amicon) at 4° C. for 45 minutes. The resultingDNA concentrate was further purified by phenol extraction and ethanolprecipitation.

[0303] Separately, 1 μg of plasmid pKK388-1 (manufactured by Clonetech)in which a SacI recognition site had been replaced with a recognitionsite for XhoI, was cleaved with the restriction enzymes NcoI and XhoIand dephosphorylated with Calf Intestine Alkaline Phosphatase[hereinafter abbreviated as CIAP; manufactured by Takara Shuzo]. Theresulting DNA was ligated by means of a ligation kit (manufactured byTakara Shuzo) with 100 ng of pKK388-1 which had also been cleaved withNcoI and XhoI and dephosphorylated as above, and the recombinant DNAthus obtained was cloned into E. coli JM 109. Plasmid pKNI5′ which hadan insertion in the normal orientation downstream from the trc promoterof pKK388-1 was thereby obtained (FIG. 2).

[0304] Plasmid pCD20-2 [Kawashima, I. et al. (1991), FEBS L. 283:199-202] contains cDNA coding for an IL-11 precursor (Pre-IL-11) andhaving a secretion signal sequence. This plasmid was cleaved with therestriction enzymes BamHI and ApaI, and a region having both the IL-11precursor (Pre-IL-11) and SV40 promoter was excised. The fragment wasligated into the BamHI and Apal sites of pBLUESCRIPT II SK+ and thenagain cleaved with the restriction enzymes XhoI and KpnI, resulting in agene which codes for a protein devoid of the N-terminus of mature IL-11(Mat-IL-11).

[0305] The resulting Mat-IL-11 fragment (devoid of its N-terminus) wasintegrated using T4 ligase into pKNI5′ which had previously been cleavedwith the restriction enzymes XhoI and KpnI and also treated with CIAP.We designated the resulting construct pKNI5IL (FIG. 3). In order toligate the specific sequence at the C-terminus of NIa with Mat-IL-11 viaAla while keeping the same reading frame, four kinds of PCR primers weresynthesized: 5′ AGGAAAAGAGTTCCTCGAGC 3′, (referred to as NSX2; sequenceID number: 5) 5′ AATTGTTCATTCCAAGCACCTGGGCCACCACCTGGC 3′, (referred toas NSJ001P; sequence ID number: 6)5′ GCCAGGTGGTGGCCCAGGTGCTTGGAATGAACAATT 3′, and (referred to as NSJ002N;sequence ID number: 7) 5′ TTGTCAGCACACCTGGGAGCTGTAGAGCTC3′. (referred toas ILSAC; sequence ID number: 8)

[0306] The first PCR reaction carried out used the pair of primers NSX2and NSJ002N, with pNS51 DNA as template to amplify a region of theinsert coding for the C-terminus of NIa (designated the CN13 region).Separately, another PCR reaction was performed using the pair of primersNSJ001P and ILSAC, with pNS51 DNA as template to amplify a region of theinsert coding for N-terminus of the IL-11 peptide (designated the 5′ ILregion). For the PCR program, a cycle of 92° C. for 1 minute, 37° C. for1 minute and 72° C. for 2 minutes was repeated 20 times followed by asingle cycle of 92° C. for 1 minute, 37° C. for 1 minute and 72° C. for30 minutes. The products obtained from each PCR procedure were extractedwith phenol and precipitated with ethanol, and then subjected to 5%polyacrylamide gel electrophoresis. A gel band containing the amplifiedDNA band with the gel was excised and the DNA was electrically recoveredfrom the gel slice using the electroelution method described above. Theresults are shown in FIG. 4.

[0307] The resulting two amplified DNA fragments were regions of theprimers NSJ001P and NSJ002N, i.e., a 3′ terminal portion of the CIN3 DNAfragment and a 5′ terminal portion of the 5′IL DNA fragment, which havepartial homology in the nucleotide sequences. Thus, if PCR is carriedout again using the primers NSX2 and ILSAC, the homologous portions willhybridize so that a fused DNA comprising portions of both genes can beobtained, the homologous portion acting as a linking part (FIG. 5).Accordingly, the recovered CIN3 DNA fragment and 5′IL DNA fragment weremixed and PCR was carried out again using only NSX2 and ILSAC asprimers. The PCR program consisted of 10 cycles of: 92° C. for 1 minute,37° C. for 1 minute and 72° C. for 2 minutes; followed by 20 cycles of:92° C. for 1 minute, 45° C. for 1 minute and 72° C. for 2 minutes;followed by a single cyle of: 92° C. for 1 minute, 55° C. for 1 minuteand 72° C. for 30 minutes. After treatment with phenol and precipitationwith ethanol, 5% polyacrylamide gel electrophoresis was performed toobtain a CNI3IL DNA fragment in which CN13 and 5′IL were fused. Themethod is shown in FIG. 5.

[0308] The CNI3IL DNA fragment was cleaved with the restriction enzymeXhoI and the resulting fragment was inserted using T4 DNA ligase intopKNI5′ which had previously been cleaved with the restriction enzymeXhoI and dephosphorylated with CIAP. The resulting plasmid was clonedinto E. coli strain JM 109. In order to select a clone in which theCNI3IL fragment had been inserted in the correct orientation, plasmidswere extracted from the resulting clones and PCR was carried out byusing the primers NSX2 and ILSAC. Using this system, only a DNA bandfrom a clone in which the fragment has been inserted in the correctorientation is detectable. Plasmid pKSUN9 in which NIa and Mat-IL-11 areligated in the same reading frame via a cleavage sequence, Gln-Ala, wasobtained (FIG. 6).

[0309] I) Expression of Ala-IL-11 in a Bacterial Cell

[0310]E. coli carrying plasmid pKSUN9 (hereinafter, abbreviated asstrain KSUN9) was cultured with shaking at 37° C. overnight in 5 ml ofLB medium containing 42 μg/ml of ampicillin. 2.5 ml of the resultingKSUN9 culture was added to 250 ml of fresh LB medium (containing thesame amount of ampicillin) and cultured with shaking at 37° C. until theOD_(600 nm) reached 1.0. Once the OD_(600 nm) had reached 1.0, IPTG wasadded to a final concentration of 0.1 mM and the mixture was culturedwith shaking at 28° C. for a further 12 hours.

[0311] A second culture was produced following exactly the sameprocedure, except that the final culture at 28° C. was performed for 36hours or longer with an IPTG concentration of 1 mM, in order to obtainmature IL-11 (which has an N-terminal Pro).

[0312] J) Western Blotting

[0313] Western blotting was performed in order to determine whetherpKSUN9 is functional in E. coli and also whether the constructedrecombinant gene is expressed.

[0314] Each of the 12 and 36 hour cultures induced with IPTG was furtherprocessed as follows. Cells were recovered from 2 ml of the culture bycentrifugation at 3,000×g at 4° C. for 5 minutes, and the pellet wassuspended in 100 μl of 20 mM sodium borate buffer (adjusted to pH 9.0with 0.1 N NaOH). The resulting suspension was treated with a sonicator(Handysonic UR-20P, manufactured by Tomy Seiko Co.) at a setting of 8for 2 minutes to disrupt the cells and then centrifuged at 10,000×g at4° C. for 10 minutes. The supernatant containing the soluble proteinfraction was recovered. Five μl of the supernatant was then run on a 12%SDS polyacrylamide electrophoresis gel following the Laemmli method[Laemmli, U.K. (1970), Nature 227: 680-685].

[0315] After electrophoresis, the gel was shaken in a transfer buffer(25 mM Tris, 192 mM glycine, 20% methanol) for 5 minutes and blottedonto a piece of PVDF membrane (Trans-Blot Transfer Medium, manufacturedby Bio-Rad Laboratories) by treatment at 15 V for 1 hour usingTrans-Blot-SD Semi-Dry Transfer Cell (manufactured by Bio-RadLaboratories). The blotted PVDF membrane was washed in PBS-Tw medium for10 minutes. The washed PVDF membrane was transferred to PBS-Twcontaining 5% Skim Milk (manufactured by Snow Brand Milk Products) at37° C. for 1 hour for blocking.

[0316] After blocking, the PVDF membrane was further washed in PBS-Twonce for 10 minutes and twice for 5 minutes, and then transferred toanti-IL-11 rabbit serum which had been diluted 10,000 fold in PBS-Tw,and incubated at 37° C. for 20 minutes. The PVDF membrane was thenwashed again with PBS-Tw, once for 10 minutes and twice for 5 minutes.

[0317] The PVDF membrane was then transferred to anti-rabbit IgG goatantibody labeled with horse radish peroxidase (manufactured by Amersham)which had been diluted 5,000 fold with PBS-Tw, and incubated at 37° C.for 20 minutes. After this time, the PVDF membrane was again washed inPBS-Tw, once for 10 minutes and 4 times, each for 5 minutes.

[0318] After this further washing, the PVDF membrane was treated withenhanced chemiluminescence (ECL) detection reagent (manufactured byAmersham), and the bands which reacted with the anti-IL-11 antibody wereshown up by bringing the PVDF membrane into contact with an X-ray filmfor a period of between 30 seconds and 5 minutes.

[0319] As a result of the above Western blotting, signal bandscorresponding to molecular weights of about 50 kDa and about 23 kDa weredetected from both the 12-hour and the 36-hour cultures. The 23 kDa bandexhibited substantially the same mobility as that of the mature IL-11used as a control. Thus, the 23 kDa signal band appeared to be IL-11which had been cleaved out of the NIa/IL-11 fusion protein by theproteolytic activity of NIa at the Gln-Ala linking sequence. It wasdeduced that the heavier band (50 kDa) was the uncleaved fusion protein.

[0320] Hereinafter, the 23 kDa protein obtained from the 12-hour culturewill be referred to as 23 kDa-ON and the 23 kDa protein obtained fromthe 36-hour culture will be referred to as 23 kDa-36hr.

[0321] K) Purification of the 23 kDa-ON and 23 kDa-36hr Proteins

[0322] The 23 kDa-ON and 23 kDa-36hr proteins were purified in order todetermine the amino acid sequences of their N-termini. 250 ml of each ofthe 12-hour and the 36-hour cultures were obtained by following the sameprocedure described in I) above. Each culture was then further processedas follows. The culture was centrifuged at 5,000×g at 4° C. for 15minutes and the pellet was suspended in 10 ml of 20 mM borate buffer (pH9.0) and disrupted with each of a French press and a sonicator. Thesoluble protein fraction was recovered as the supernatant aftercentrifugation at 15,000×g at 4° C. for 30 minutes.

[0323] The resulting soluble protein fraction was then subjected to weakion exchange column chromatography using FPLC manufactured by Pharmaciausing the following conditions: Column: CM Toyopearl Pack 650 M (2.2 ×20 cm, manufactured by Toso) Elution buffers: A = 10 mM boricacid-sodium hydroxide (pH 9.0), 13 mM potassium chloride B = 10 mM boricacid-sodium hydroxide (pH 9.0), 13 mM potassium chloride, 400 mM sodiumchloride Flow rate: 2.5 ml/min Fraction volume: 5 ml/tube

[0324] The concentration gradient used was a linear gradient from eluentA to eluent B over a period of 120 minutes.

[0325] Each eluted fraction was subsequently subjected to anenzyme-linked immunosorbent assay (ELISA), to identity fractionscontaining IL-11.

[0326] The ELISA was performed as follows. Each well of a 96well-immunoplate (Maxisoap; manufactured by Nunc) was loaded with 100 μlof 50 mM sodium carbonate buffer (pH 9.6) containing 1 μg/ml ofanti-IL-11 mouse monoclonal antibody and the plate was then incubated at37° C. for 1 hour. After this time, each well was washed four times withPBS-T medium (PBS containing 0.1% Tween 20).

[0327] Each of the FPLC fractions obtained above was diluted 100 foldwith PBS-T and loaded into the wells in aliquots of 100 μl /well. Theplate was incubated at 37° C. for 1 hour, the wells were again washedwith PBS-T, and then each well was loaded with 100 μl of anti-IL-11rabbit IgG diluted with PBS-T to a final concentration of 1 μg/ml.

[0328] The plate was further incubated at 37° C. for 1 hour and washedwith PBS-T, and then each well was loaded with 100 μl of alkalinephosphatase-labeled goat anti-rabbit IgG antibody (manufactured by GibcoBethesda Research Laboratories) which had been diluted 3,000 fold inPBS-T. The plate was again incubated at 37° C. for 1 hour and thenwashed with PBS-T. Each well was then loaded with 100 μl of alkalinephosphatase substrate solution. The plate was incubated at roomtemperature for a further 30 minutes to 1 hour, after which time thecoloring of each well was measured as absorbance at 405 nm to identifythe fraction(s) of interest.

[0329] The fractions from the FPLC procedure identified by the aboveELISA procedure as containing a substance reacting with rabbitanti-IL-11 antibody (fraction nos. 19 to 25) were pooled, concentrated100-fold with Centprep-10 (manufactured by Amicon), and then run on a12% SDS polyacrylamide electrophoresis gel in accordance with theLaemmli method (supra). The gel was then electro-blotted onto a piece ofPVDF membrane in a manner similar to that used in the above in theWestern blotting procedure, but using the Problot membrane (manufacturedby Applied Biosystems) as the PVDF membrane.

[0330] After blotting, the membrane was thoroughly washed withredistilled water, stained with Coomassie Brilliant Blue R-250,destained with 50% methanol, and then the band containing the proteinwhich reacted with anti-IL-11 antibody in the Western blot was excised.

[0331] The amino acid sequence of the N-terminus of the protein wasanalyzed using a protein sequencer (manufactured by Applied Biosystems).The N-terminal sequence of the band from 23 kDa-ON which reacted withthe anti-IL-11 antibody was determined to be:

[0332] Ala-Pro-Gly-Pro-Pro-Pro-Gly-(sequence ID No. 9)

[0333] This sequence corresponds to the amino acid sequence −1 to +6 ofthe amino acid sequence of the mature IL-11 protein. Based on thisfinding, it could be deduced that the 23 kDa protein obtained from the12-hour culture and which reacted with anti-IL-11 antibody wasAla-IL-11. Accordingly, it seemed apparent that this protein wasgenerated by the proteolytic activity of NIa cleaving the NIa/IL-11fusion protein at the Gln-Ala site in the specific cleavage sequence.

[0334] The N-terminal sequence of the band from 23 kDa-36 hr whichreacted with the anti-IL-11 antibody was determined to be:

[0335] Pro-Gly-Pro-Pro-Pro-Gly-Pro-(sequence ID No. 10)

[0336] This sequence corresponds to the amino acid sequence +1 to +7 ofmature IL-11. Accordingly, we were able to reach the followingconclusions.

[0337] Following induction with IPTG, the NIa/IL-11 fusion protein wasexpressed in E. coli.

[0338] By 12 hours culturing subsequent to induction, the expressedNIa/IL-11 fusion protein was cleaved at the Gln-Ala peptide bond in thespecific cleavage sequence by the protease activity of NIa.

[0339] After cleavage of the peptide bond with NIa, mature IL-11 buthaving an extra Ala on its N-terminus was expressed in E. coli.

[0340] By culturing for a further 24 hours after the expression ofAla-IL-11 had been established, Ala-IL-11 matured to IL-11 in which theAla residue which had been present at the N-terminus of Ala-IL-11 wasdeleted.

[0341] Based on the above findings, it was concluded that the 23 kDaprotein obtained after culturing for 36 hours was a mature type of IL-11whose N-terminus was Pro.

[0342] Therefore, it was established that IL-11 could be expressed as afusion protein with NIa and that the activity of NIa could cleave aspecific linker sequence containing Gln-Ala to afford Ala-IL-11.Continued culture was then able to afford mature IL-11 wherein thealanine residue had been deleted to expose a proline N-terminal by afactor present in E. coli.

[0343] In order to ensure that the expressed IL-11 and Ala-IL-11 werebiologically active, adipogenesis inhibitory factor (AGIF) activity wasdetermined as an index. The fact that IL-11 has adipogenesis inhibitoryactivity has previously been demonstrated [Kawashima, I. et al. (1991),FEBS L. 283: 199-202].

[0344] L) Measuring Inhibitory Effects on the Morphological Changes from3T3-L1 Cells to Adipocytes

[0345] The method for determining the AGIF activity used in the presentinvention is as follows. Mouse embryonic fibroblast cell line 3T3-L1[Green, H. and Kehinde, O. (1974), Cell 1: 113-116] purchased from ATCCis used. The cells are in all cases cultured in a mixed humid atmosphereof 10% CO₂-90% air at 37° C. and subcultured with medium A. Induction ofadipogenic differentiation is carried out following the proceduredescribed by Rubin et al. [Rubin, C. S. et al. (1978), J. Biol. Chem.253: 7570-7578].

[0346] 3T3-L1 cells are suspended in medium A to a density of 1.0×10⁴cells/ml, seeded onto a 48 well multicluster dish (manufactured byCoaster, 0.5 ml/well), and then cultured. After 3 days of culturing, thecells reach confluence. The medium is then replaced with fresh medium Aand, after culturing for a further 2 days, the medium is replaced withan adipogenesis induction medium, medium B, together with thesimultaneous addition of 0.5 ml of the test sample. The medium isreplaced with fresh medium B and a fresh sample every two days.

[0347] Instead of medium B, adipocyte maintaining medium, medium C isused to replace exhausted medium in the wells, starting at varying timesfor different wells between days 4 and 7 after the first test sample isadded.

[0348] After culturing in medium C for 2 days, the cells are fixed with5% formaldehyde, and any fat particles which have accumulated in thecells and cell nuclei are stained with Oil Red 0 and hematoxylin,respectively. A micrograph is taken and the number of nuclei and thenumber of cells which have accumulated stained fat particles arecounted. The adipogenetic ratio (AR) is calculated according to thefollowing equation:${{AR}(\%)} = {100 \times \frac{{number}\quad {of}\quad {cells}\quad {accumulating}\quad {fat}\quad {particles}}{{total}\quad {number}\quad {of}\quad {nuclei}}}$

[0349] Fixation of the cells and staining with Oil Red 0 and hematoxylinare carried out in accordance with the procedures described by YoshioMitomo and Shojiro Takayama in “Lectures on Clinical Testing” Vol. 12,“Pathology” (1982), published by Ishiyaku Shuppan.

[0350] M) Method for the Determination of Lipoprotein Linase InhibitoryActivity

[0351] The determination is carried out in accordance with the methoddescribed by Beutler et al. [Beutler, B. A. et al. (1985), J. Immonol.135: 3972-3977]. Adipogenically differentiated 3T3-L1 cells are preparedas described in L) above, except that no test sample is added whendifferentiation into adipocytes is induced. Instead, the test sample isadded together with fresh medium C and the cells are cultured for 18hours.

[0352] After this time, the medium is removed and the cells are washedtwice with PBS(−) (phosphate-buffered saline, manufactured by NissuiSeiyaku) and each well is then loaded with 300 μl of medium D andcultured for a further 1 hour. 100-μl aliquots of each of the culturesupernatants are taken for use in measuring lipoprotein lipase (LPL)activity, which is measured in triplicate for each sample to obtain anaverage.

[0353] LPL activity is measured as described by Nilsson-Ehle and Schotz[Nilsson-Ehle, P. and Schotz, M. C. (1976), J. Lipid Res. 17: 536-541].The aliquots of supernatant obtained above are each mixed with an equalvolume of LPL substrate solution and allowed to react at 37° C. for 120minutes. The reaction is stopped by adding 1.05 ml of 0.1 M potassiumcarbonate buffer (adjusted to pH 10.5 with 0.1 M potassium borate) and3.25 ml of a mixture of methanol:chloroform:heptane [141:125:100 (v/v)]with vigorous stirring. The mixture is then centrifuged at 3,000×g for15 minutes at room temperature. The H content of the water-methanolphase is counted with a liquid scintillation counter.

[0354] One unit of LPL activity is defined as being that activity whichgenerates 1 μmol of fatty acid per minute. The 13 mM glyceroltri[9,10(n)-³H]oleate in the substrate solution is prepared by dilutingglycerol tri-[9,10(n)-³H]oleate (37.0 GBq/mol), manufactured by Amershamwith triolein (manufactured by Sigma), followed by purification on asilica gel column chromatography.

[0355] The following Examples relate to the glutathione reducing proteinembodiment of the present invention.

EXAMPLE 1

[0356] Extraction of Poly(A)^(±) RNA from KM-102 Cells

[0357] KM-102 cells were cultured in 36 plastic, 15 cm diameter, culturedishes with Iscove's modified minimum essential medium(Boeringer-Mannheim) containing 10% fetal bovine serum. After growingthe cells to confluence, phorbol myristyl acetate (PMA) and calciumionophore A23187 (Sigma) were added to final concentrations of 10 ng/mland 0.2 μM, respectively, and culturing was continued at 37° C. Lots of12 dishes were harvested 3, 6 and 14 hours later, and each dish wasseparately dissolved in guanidine thiocyanate solution and the liquidphase was collected.

[0358] Isolation of poly(A)⁺ RNA was basically performed as described in“Molecular Cloning—A Laboratory Manual” [Maniatis, T. et al. (1982) pp.196-198]. The following provides a detailed description of theprocedure.

[0359] Each recovered liquid phase was individually treated as follows.The liquid was repeatedly drawn up and discharged from a 10 ml syringebarrel equipped with a 21 G syringe needle. 3 ml of a solution of 5.7 MCsCl-0.1 M EDTA (pH 7.5) was added in advance to a Polyaromar centrifugetube matching the size of a rotor bucket of an RPS40-T centrifuge(Hitachi Koki). The cell preparation was then overlaid on the solutionin the tube until the tube was full.

[0360] After centrifuging at 30,000 rpm for 18 hours at 20° C., theresulting pellet was suspended in 400 μl of distilled water followed byethanol precipitation. The resulting pellet was dissolved in 400 μl ofdistilled water and added to an equal volume of chloroform/1-butanolmixture (4:1 v/v) with stirring and the aqueous layer was collected bycentrifugal separation. Ethanol precipitation was performed once again,and the resulting pellet was dissolved in 600 μl of distilled water toobtain whole RNA. About 4.5 mg of whole RNA was obtained from each ofthe pooled PMA/A23187-stimulated samples from 3, 6 and 14 hours.

[0361] 600 μg of each of the three types of whole RNA obtained in thismanner were pooled and subjected to oligo(dT) cellulose columnchromatography to purify the poly(A)⁺ RNA.

[0362] The whole RNA was dissolved in adsorption buffer, and heated at65° C. for 5 minutes. The resulting solution was applied to an oligo(dT)cellulose column (Pharmacia, type 7) loaded with adsorption buffer, andeluted with eluting solution to recover 100 μg of poly(A)⁺ RNA.

EXAMPLE 2

[0363] Preparation of a cDNA Library

[0364] A cDNA library was prepared by the Okayama-Berg method.

[0365] 5 μg of poly(A) ⁺RNA and 24 units of reverse transcriptase(Seikagaku Corp.) were allowed to react at 42° C. for 1 hour in 20 μl ofreverse transcriptase reaction solution.

[0366] The reaction was stopped by the addition of 2 μl of 0.25 M EDTAand 1 μl of 10% SDS, and the solution was then deproteinized with 20 μlof a phenol/chloroform mixture (1:1 v/v). Following centrifugation toremove the proteinaceous fraction, 20 μl of 4 M ammonium acetate and 80μl of ethanol were added to the supernatant which was then cooled at−70° C. for 15 minutes. After this time, the precipitate was collectedby centrifugal separation, washed with 75% ethanol and then dried underreduced pressure.

[0367] The dried precipitate was dissolved in 15 μl of terminaltransferase reaction solution and warmed at 37° C. for 3 minutes. At theend of this time, 18 units of terminal deoxynucleotidyl transferase(Pharmacia) were added to the reaction solution and allowed to react for5 minutes. 1 μl of 0.25 M EDTA and 0.5 μl of 10% SDS were added to stopthe reaction and the solution was then deproteinized withphenol-chloroform (as described above) and centrifuged to remove theproteinaceous fraction. The supernatant was collected and thoroughlymixed with 15 μl of 4 M ammonium acetate and 60-1 of ethanol. Thismixture was cooled at −70° C. for 15 minutes and the precipitate wascollected by centrifugation.

[0368] The resulting pellet was dissolved in 10 μl of restriction enzymebuffer, and 2.5 units of restriction enzyme HindIII were added to theresulting solution which was allowed to stand at 37° C. for 1 hour toeffect digestion.

[0369] The reaction solution was then deproteinized withphenol-chloroform followed by ethanol precipitation, and the supernatantwas cooled at −70° C. for 15 minutes. The resulting precipitate wascollected by centrifugation and dissolved in 10 μl of TE buffer [10 mMTris-HCl (pH 7.5) and 1 mM EDTA]. 1 μl of the resulting solution wasmade up to 10 μl of a reaction solution containing 10 mM Tris-HCl (pH7.5), 1 mM EDTA, 100 mM NaCl and 10 ng of oligo(dG)-tailed linker DNA[3′-oligo(dG)-tailed pL-1 HindIII linker, Pharmacia], followed byheating at 65° C. for 5 minutes and then warming at 42° C. for 30minutes. The reaction mixture was cooled over ice water, followed by theaddition thereto of 10 μl of 10× ligase buffer, 78 μl of distilled waterand 8 units of T4 DNA ligase. The reaction solution was then keptovernight at 12° C.

[0370] The following day, the reaction mixture was combined with 10 μlof 1 M KCl, 1 unit of ribonuclease H, 33 units of DNA polymerase I, 4units of T4 DNA ligase, 0.5 μl of dNTP solution (20 mM DATP, 20 mM dCTP,20 mM dGTP and 20 mM dTTP) and 0.1 μl of 50 μg/ml bovine serum albumin(BSA), and the resulting mixture was warmed first at 12° C. for 1 hourand then at 25° C. for 1 hour. After this time, the reaction solutionwas diluted five-fold with distilled water and was then immediately usedto transform E. coli DH5 α using the Hanahan method [Hanahan, D. (1983)J. Mol. Biol. 166, 557-580] thereby to prepare a cDNA library of KM-102cells.

EXAMPLE 3

[0371] Preparation of an Oligonucleotide Probe

[0372] Based on the AUUUA sequence in the 3′ non-translated region ofthe mRNA of cytokines, the 15-base oligonucleotide 5′-TAAATAAATAAATAA-3′(sequence ID number 13), designated ATT-3, was chemically synthesized.Synthesis was performed using the 380B automatic DNA synthesizer(Perkin-Elmer Japan Applied Biosystems) following the directionssupplied in the accompanying manual. The method employed was thephosphoamidite method described by Caruthers et al. [Matteucci, M. D.and Caruthers, M. H. (1981) J. Am. Chem. Soc. 103, 3185-3191]. Aftersynthesis of the 15-mer, the resulting oligonuculeotide was severed fromthe support and the protecting groups were removed. The resultingoligonucleotide solution was lyophilized to form a powder which was thendissolved in distilled water and stored frozen at −20° C. until the timeof use.

EXAMPLE 4

[0373] Screening the cDNA Library

[0374] 6,500 colonies generated from the cDNA library prepared inExample 2 above were fixed on a nitrocellulose filter in accordance withthe method described by Grunstein and Hogness [Grunstein, M. andHogness, D. S. (1975) Proc. Natl. Acad. Sci. USA 72, 3961-3965]. TheATT-3 probe prepared in Example 3 was 5′-labelled with ³²P followingstandard procedures (see “Molecular Cloning—A Laboratory Manual”), andthe labelled probe was used for colony hybridization.

[0375] Pre-hybridization was performed at 37° C. for 3 hours in thefollowing: 6×SSC, 1× Denhardt solution, 0.25% SDS, 0.05% sodiumpyrophosphate and 100 μg/ml of denatured salmon sperm DNA. Hybridizationwas then performed overnight at 31° C. in the following: 6×SSC, 1×Denhardt solution, 17 μg/ml of yeast tRNA and 0.05% sodium pyrophosphatecontaining the ³²P-labeled probe ATTT-3.

[0376] On the following day, the nitrocellulose filter was washed atroom temperature for 2 hours with a 6×SSC solution containing 0.05%sodium pyrophosphate. Subsequent autoradiography revealed 33 positiveclones.

[0377] The plasmid DNA was extracted from the positive clones byfollowing standard procedures. Several clones were then selected atrandom and their partial cDNA nucleotide sequences were determined bythe dideoxy method. These partial sequences were then examined forhomology with nucleotide sequences registered in the EMBL or GenBankdatabases via a personal computer and it was established that some ofthe partial sequences of clones detected by ATT-3 had homology withparts of the Alu repeat [Schmid, C. W. and Jelinek, W. R. (1982) Science216, 1065-1070].

[0378] A DNA fragment containing the Alu repeat sequence was preparedfrom human genome DNA and labeled with ³²P, following standardprocedures. This labeled DNA was used as a probe in colony hybridizationusing the 33 clones identified above, and it became clear that 12 of theclones possessed the Alu repeat. The length of the cDNA insert of eachof the remaining 21 clones was determined, and it was established thatthe length was variable over a range of 50 to 3,600 bases.

[0379] Restriction enzyme mapping was performed on the cDNA inserts ofthe remaining 21 clones, and partial nucleotide sequences weredetermined as above. These partial sequences were then examined as abovefor homology with nucleotide sequences registered in the EMBL or GenBankdatabases via a personal computer, and those clones having novelsequences were selected.

EXAMPLE 5

[0380] Northern Hybridization of Clone No. 31

[0381] One of the clones, clone no. 31 (designated pcD-31) had a cDNAinsert of about 560 bp. A PstI-AatI fragment (292 bp) was obtained fromthe cDNA insert of pcD-31 and was labeled with ³²P for use as a probe ina Northern blot procedure using poly(A)⁺ RNA prepared from KM-102 cells,following a procedure similar to that of Example 1. This hybridizationwas used to determine the length of the naturally occurring mRNA whichcorresponds to the insert of pcD-31.

[0382] The procedure of the Northern hybridization was as follows. 5.5μg of poly(A)⁺ RNA was prepared from KM-102 cells and incubated at 50°C. for 1 hour in a mixture of 1 M glyoxal, 50% dimethyl sulfoxide (DMSO)and 0.01 M disodium hydrogen phosphate (pH 7.0). At the end of thistime, 4 μl of electrophoresis pigment were added to the incubatedmixture which was then electrophoresed on a 1% agarose gel in 1× TAE.

[0383] Following the electrophoresis, the RNA on the agarose gel wastransferred overnight onto a nylon membrane filter (Bio Rad, Zeta-Probe)using 20×SSC using the capillary transfer method (see “MolecularCloning—A Laboratory Manual”). After transferrence, the filter wasgently washed with 2×SSC, air dried, and then additionally dried at 80°C. for 2 hours to fix the mRNA.

[0384] The PstI-AatI fragment of pcD-31 was labeled with ³²P using theMultiprime DNA Labeling System (Amersham).

[0385] Pre-hybridization was performed on the filter for 3 hours at 37°C. in a solution containing 5×SSCP, 2.5× Denhardt solution, 50%formamide, 10 mM disodium hydrogen phosphate (pH 7.0), 0.5% SDS and 100μg/ml of denatured salmon sperm DNA.

[0386] Hybridization was then performed on the filter overnight at 42°C. in a solution containing the ³²P-labeled probe, 5×SSCP, 1× Denhardtsolution, 50′ formamide, 10 mM disodium hydrogen phosphate (pH 7.0),0.1% SDS and 100 μg/ml of denatured salmon sperm DNA. The following day,the filter first was washed for 1 hour at 37° C. with a solutioncontaining 50% formamide, 5×SSC and 0.1% SDS, then washed for 2 hours atthe same temperature with a solution containing 409 formamide, 5×SSC and0.1% SDS, and finally washed at room temperature for 15 minutes with asolution of 2×SSC. Subsequent autoradiography showed that the cDNAinsert of clone pcD-31 is not full length, and that the full length ofthe corresponding mRNA is 3.9 kb (determined from a calibration curvebased on molecular weight markers).

EXAMPLE 6

[0387] Preparing a Fresh Library for Screening Clone pcD-31 cDNA

[0388] A fresh cDNA library was prepared using the cDNA Synthesis SystemPlus and cDNA Cloning System (λgt10, adapter method, supplied byAmersham).

[0389] 5 μg of poly(A)⁺ RNA extracted from KM-102 cells (following aprocedure similar to that of Example 1) and 100 units of reversetranscriptase were reacted at 42° C. for 40 minutes in 50 μl of reversetranscriptase reaction solution. After this time, 20 μCi of [α-³²P]dCTP,93.5 μl of second strand buffer, 4 units of ribonuclease H and 115 unitsof DNA polymerase I (all provided with the kit) were added to thereaction solution which was then first incubated at 12° C. for 1 hour,then incubated at 22° C. for 1 hour and finally heated at 70° C. for 10minutes. After this heating treatment, 10 units of T4 DNA polymerase(provided with the kit) were added to the reaction solution and allowedto react at 37° C. for 10 minutes.

[0390] The reaction mixture was then subjected to phenol-chloroformdeproteinization. The reaction solution was centrifuged and thesupernatant was collected and mixed well with 250 μl of 4M ammoniumacetate and 1 ml of ethanol. The mixture was cooled overnight at −20° C.and the precipitate was collected by centrifugation. The resultingpellet was dissolved in 30 μl of sterile water. 10 μl of the resultingsolution were removed and added to a mixture of 2 μl of ligase/kinasebuffer, 250 μmole of EcoRI adapter and 5 units of T4 DNA ligase (allprovided with the kit) and the resulting mixture was incubated overnightat 15° C.

[0391] The whole of the reaction solution was then applied to the sizefractionation column provided with the kit in order to remove the EcoRIadapter. The reaction solution was collected in 120 μl aliquots and eachaliquot was mixed with 200 μl of 0.25× TE buffer. The 10th to 17thfractions were pooled and concentrated with butanol to a total volume of120 μl.

[0392] The whole of concentrated prearation was then mixed with 55 μl ofsterile water, 20 μl of ligase/kinase buffer and 40 units of T4polynucleotide kinase (all provided with the kit), and the resultingmixture was incubated at 37° C. for 30 minutes. After this time, thereaction mixture was deproteinized by the phenol-chloroform method threetimes and then precipitated with ethanol and cooled overnight at −20° C.The resulting precipitate was collected by centrifugation and dissolvedin 10 μl of sterile water to provide the cDNA sample.

[0393] 1 μg of the EcoRI arm of λgt10, 1 μl of ligase/kinase buffer and2.5 units of T4 DNA ligase (all provided with the kit) were added toeither 2 μl of the cDNA sample, followed by incubation overnight at 15°C. A sample containing 4 μl of the cDNA sample was prepared in the sameway. Each sample was then further treated as follows. The whole of theresulting reaction solution was first added to 10 μl of Extract A(provided with kit) and the resulting mixture was then added to 15 μl ofExtract B (provided with kit), and the resulting mixture was incubatedat 20° C. for 20 minutes to allow the in vitro packaging reaction totake place.

[0394] After this time, 470 μl of SM buffer were added to the reactionsolution which was then stored at 4° C. E. coli strain NM514 treatedwith 10 mM MgSO₄ was then infected with the stored solution to create aλgt10 library of KM-102 cDNA.

EXAMPLE 7

[0395] Screening the cDNA Library

[0396] 2×10⁵ plaques obtained from the combined cDNA libraries preparedin Example 6 were fixed to nylon filters (Hybond N, Amersham) by thefollowing procedure.

[0397] Infected E. coli prepared in Example 6 were cultured on ten 9 cmplates containing solid LB medium so that between 1 and 2×10⁴ plaqueswere formed per plate. The plaques were transferred onto the plate bygently pressing the nylon filter onto the plate. An 18 G syringe needlewas then used to puncture the filter and mark the gel at 3 locations forreference. The filter was then allowed to stand at 4° C. for 5 to 10minutes and was then peeled off and dipped in an alkaline solution (0.1N NaOH, 1.5 M NaCl) for 20 seconds. The filter was then transferred to aneutral solution [0.2 M Tris-HCl (pH 7.5), 2×SSCP] for a period ofbetween 20 seconds and 1 minute, and was next air-dried at roomtemperature for 2 hours. Finally, the filter was dried at 80° C. for 2hours.

EXAMPLE 8

[0398] Probe Preparation and Hybridization

[0399]³²P-Labeled probes were created from from pcD-31 (obtained inExample 4) by labeling the PstI-AatI fragment and the EcoT221-AatIfragment (223 bp) from pcD-31 using the Multiprime DNA Labelling System.Plaque hybridization was performed on the filter obtained in Example 7using the above probes.

[0400] Pre-hybridization was performed by placing the filter in a bathof 50% formamide, 5×SSCP, 2.5× Denhardt solution, 0.01 M disodiumhydrogen phosphate (pH 7.0), 0.5% SDS and 100 μg/ml of denatured salmonsperm DNA and incubating at 37° C. for 2 hours.

[0401] Hybridization was then performed by placing the filter in areaction solution containing the ³²P labeled probes prepared above and50% formamide, 5×SSCP, 1× Denhardt solution, 0.01 M disodium hydrogenphosphate (pH 7.0), 0.1% SDS and 100 μg/ml of denatured salmon spermDNA, and incubating overnight at 37° C.

[0402] The following day, the filter was first washed at roomtemperature for 3 hours with a solution containing 50% formamide, 5×SSCand 0.1% SDS, and then washed at room temperature for 5 minutes with2×SSC. Autoradiography showed 80 positive clones obtained in thisprimary screen.

[0403] Using the clones identified as positive each time, the proceduresof Examples 7 and 8 were repeated a further three times (quaternaryscreening), and a total of 17 positive clones were ultimately obtained.The cDNA was isolated from each of the 17 clones and the inserts werecut out with EcoRI. The length of each cDNA insert was investigated byagarose gel electrophoresis and clone no. 31-7 was isolated, which had acDNA insert of 3.9 kbp, corresponding to the complete length of theoriginal mRNA.

EXAMPLE 9

[0404] Restriction Mapping of Clone No. 31-7

[0405] Clone no. 31-7 was digested with EcoRI to isolate and purify the3.9 kb fragment containing the cDNA insert. This fragment was theninserted into pUC18 using T4 DNA ligase. E. coli DH5α was transformedwith this new plasmid. Transformed cells were selected by theirresistance to ampicillin and clone pUCKM31-7 having a 3.9 kbp cDNAinsert was identified by digesting the DNA with EcoRI and subjecting thecleaved DNA to agarose gel electrophoresis.

[0406] pUCKM31-7 was cleaved with each of the restriction enzymesHindIII, SacI, XbaI, SmaI, BgIII, EcoT22I and AatI, or pairs thereof.Agarose gel electrophoresis was performed on the resulting fragments andthe length of each fragment was measured using the λHindIII/φX174HaeIIImarker as an indicator. The resulting restriction map is shown in FIG.7.

EXAMPLE 10

[0407] Sequence Determination of Clone No. 31-7

[0408] The entire nucleotide sequence of the cDNA insert of pUCKM31-7was determined by the dideoxy method using an M13 phage. In addition, aportion of the sequence was analyzed with the 373A DNA Sequencer(Perkin-Elmer Japan Applied Biosystems). The resulting nucleotidesequence is ID number 11 in the accompanying Sequence Listing.

[0409] The cDNA insert of pUCKM31-7 is 3815 bases long, and clearly hasan open reading frame composed of 549 amino acids, starting withmethionine. A poly(A) tail is apparently absent. A comparison of thebase sequence of the 3′ terminal of the insert of pcD-31 with thesequence of clone pUCKM31-7 reveals that the insert of pUCKM31-7 is onlymissing the poly(A) tail portion (FIG. 8), and nothing else.

[0410] The EMBL and GenBank nucleotide databases and the NBRF andSWISS-PROT databases were accessed in order to compare the base andamino acid sequences, respectively. The closest match which wasdiscovered was a 35.3% homology of the peptide sequence with humanglutathione reductase. Accordingly, it was concluded that the ORF of thecDNA insert of pUCKM31-7 clearly encodes a novel polypeptide. This novelpolypeptide is shown as sequence ID number 12 in the accompanyingSequence Listing.

EXAMPLE 11

[0411] Expression and Purification of the Novel Polypeptide

[0412] Construction of a High Expression Vector and Expression in COS-1Cells

[0413] pUCKM31-7 was digested with HindIII and the 3003 bp fragmentcontaining the cDNA insert was isolated and purified following standardprocedures. The terminals of the resulting fragment were blunted using aDNA blunting kit (Takara Shuzo).

[0414] Meanwhile, the high expression vector pcDL-SRα296 [Takabe, Y. etal. (1988) Mol. Cell. Biol. 8, 466-472] was digested with PstI and KpnIand the terminals were blunted using a DNA blunting kit. The bluntedinsert was then ligated into the blunted plasmid in a reaction using T4DNA ligase. E. coli was then transformed with the resulting DNA by thecalcium chloride method, and the resulting Amp^(R) transformants wereselected and the plasmid DNA retained by the organisms was analyzed.

[0415] Specifically, a strain in which the direction of cDNAtranscription was identical to the direction of the SRα promoter wasselected by digestion of the plasmid with HindIII and BglII followed byagarose gel electrophoresis to locate an 800 bp fragment, and theplasmid which was selected was designated pSRα31-7 (FIG. 9). The SRαpromoter comprises the SV40 initial promoter and the R-U5 sequence ofthe long terminal repeat (LTR) of HTLV-1, and has promoter activitywhich is 10 to 100 times stronger than the SV40 initial promoter alone.

[0416] Next, COS-1 cells were transfected with the resulting plasmidpSRα31-7. Transfection of COS-1 cells was performed by electroporationusing the GTE-1 gene introduction device (Shimadzu).

[0417] COS-1 cells were grown to semi-confluence over the bottoms ofseven 150 cm³ flasks, each containing 25 ml DMEM (containing 10% fetalbovine serum). The cultures were then collected and each was treatedwith 3 ml of Trypsin-EDTA solution (10× solution available from Sigma)and allowed to stand at room temperature until the cells had separated.1 ml of inactivated fetal bovine serum and 9 ml of fresh trypsin-EDTAsolution were then added and the cells were collected by centrifugation.The collected cells were then washed twice with PBS(−) buffer andsuspended in PBS(−) buffer to a density of 6×10⁷ cells/ml.

[0418] Meanwhile, plasmid DNA was prepared by the cesium chloride methodand made up to 200 μg/ml in PBS(−) buffer.

[0419] 20 μl of each of the above-mentioned PBS(−) preparations of cellsand plasmid were mixed and the resulting mixture was placed in a chambercontaining electrodes spaced apart at an interval of 2 mm. Two 600Vpulses, each for a duration of 30 μsec were then applied to the mixtureat an interval of 1 second.

[0420] The electrode chamber was cooled at 4° C. for 5 minutes and thenthe cell-DNA mixture inside was mixed with 10 ml of DMEM containing 100fetal bovine serum. This mixture was transferred to a Petri Plate andcultured overnight at 37° C. in a 50% CO₂ atmosphere. The following day,the culture supernatant was discarded, the cells were washed withserum-free DMEM and then suspended in 10 ml of DMEM and cultured at 37°C. for 3 days. After this time, the cell supernatant was harvested.

[0421] Culture supernatant was also harvested from the negative control.The negative control used the plasmid pcDL-SRα296 containing no cDNAinsert, but was otherwise prepared in similar manner to the testculture.

[0422] 1 ml of each of the culture supernatants of the negative controland the test culture were separately processed as follows. Thesupernatant was first treated with trichloroacetic acid (TCA) toprecipitate protein, and the precipitate was collected by centrifugalseparation. The resulting precipitate was washed with ice-cooled acetoneand air-dried and then dissolved in SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) sample buffer containing 2-mercaptoethanol.SDS-PAGE was then performed on a 12.5% gel under reducing conditions.

[0423] Silver staining using the silver stain reagent “Daiichi” (DaiichiChemical Detection) was performed on the bands followingelectrophoresis. Several specific bands (molecular weight: about 60,000)from the culture supernatant of the test sample were stained.

[0424] Since the molecular weight of the polypeptide encoded in pSRα31-7is about 60,000, and it was also deduced from the amino acid sequencethat post-translational modification to add saccharide side chains wasunlikely, it was concluded that these several specific bandscorresponded to the polypeptide encoded by the cDNA of pSRα31-7.

EXAMPLE 12

[0425] Preparation of a High Expression Plasmid for COS-1 Cells

[0426] The next step was to verify that the several specific 60 kDabands identified in Example 11 are the same as the polypeptide encodedby the insert of pSRα31-7. It was also desired to determine theN-terminal amino acid sequence of this polypeptide. Accordingly, a clonewas prepared wherein an extra six His residues were encoded for theC-terminal of the polypeptide encoded by the pSRα31-7 insert,immediately before the stop codon. Histidine residues have a highaffinity for Ni²⁺and the objective was to express a polypeptide having ahistidine hexomer (6× His), which could be purified using an affinityresin column charged with NI²⁺.

[0427] First, a 66 base oligonucleotide5′-CTAGCGCTCTGGGGCAAGCATCCTCCAGGCTGGCTGCCACCACCACCACCACCACTGATCTAGACT-3′(sequence ID No. 14) and the complementary 66 base strand weresynthesized and purified using an Automated DNA Synthesizer 394(Perkin-Elmer Japan Applied Biosystems). Both oligonucleotidepreparations were mixed and incubated at 70° C. for 3 minutes and thenwere additionally warmed at 37° C. for 30 minutes to allow annealing.Subsequently, the terminals were phosphorylated using T4 polynucleotidekinase.

[0428] The resulting double-stranded (ds) fragment was ligated using T4DNA ligase into pUCKM31-7 which had previously been digested withEco47III. The construct is shown in FIG. 10. E. coli DH5α wastransformed with this DNA by the calcium chloride method and theresulting transformed strains were selected and screened to obtainpUCKM31-7His. It was confirmed that there were no abnormalities in theportion of pUCKM31-7His where the fragment was inserted by analyzing aportion of the relevant base sequence of this pUCKM31-7His.

[0429] A high-expression plasmid for COS-1 cells was then prepared bysubcloning the insert of pUCKM31-7His into pcDL-SRα296.

[0430] pUCKM31-7His was digested with XbaI and HindIII, the fragmentswere purified and the terminals of the fragments were blunted using 1unit of Klenow fragment in the presence of 2 mM DATP, 2 mM dCTP, 2 mMdGTP, 2 mM dTTP, 50 mM Tris-HCl (pH 7.2), 10 mM MgSO₄, 0.1 mMdithiothreitol and 50 μg/ml of BSA.

[0431] Meanwhile, the high expression vector pcDL-SRα296 was digestedwith PstI and KpnI and blunt-ended with a DNA blunting kit. The bluntedfragment was then ligated into the blunted plasmid using T4 DNA ligase.The resulting plasmid was then used to transform E. coli DH5α.Transformants were then selected and screened. A strain in which thedirection of cDNA transcription was identical to the direction of theSRα promoter was selected, and the plasmid of this strain was designatedpSRα31-7His. COS-1 cells were transfected with the resulting plasmidpSRα31-7His and serum-free supernatant was obtained in a manner similarto that described in Example 11.

EXAMPLE 13

[0432] Purification and N-Terminal Amino Acid Sequence Analysis

[0433] 600 ml of the supernatant obtained in Example 12 were subject todialysis against 17 volumes of dialysis buffer at 4° C. for 15 hours.The buffer was replaced with a further 17 volumes of dialysis buffer anddialysis was continued at 4° C. for an additional 4 hours.

[0434] The dialyzed preparation was then subjected to affinitychromatography using FPLC (Fast Protein Polynucleotide LiquidChromatography—Pharmacia) under the following conditions:

[0435] Column: 20 ml of ProBond™ Resin (Invitrogen) filled into XK16/20(φ2.0×20 cm, Pharmacia)

[0436] Elution buffer:

[0437] A) 20 mM phosphate buffer (pH 7.8) containing 200 mM imidazole,0.5 M NaCl

[0438] B) 20 mM phosphate buffer (pH 7.8) containing 300 mM imidazole,0.5 M NaCl

[0439] Flow rate: 1 ml/min

[0440] Fraction solution: 5 ml/tube

[0441] Elution conditions: After recovering 4 fractions with elutionbuffer A), 16 fractions were recovered with elution buffer B), and thefractions were numbered in order from 1 to 20.

[0442] 300 μl of each of the resulting fraction samples were taken andseparately treated subjected to TCA precipitation treatment and theresulting precipitate was prepared and subjected to SDS-PAGE using a12.5% gel under reducing conditions as before. Bands were detected bythe silver stain method and three bands were detected centering aroundfraction no. 10. The existence of 3 bands indicates that the pSRα31-7Hisinsert encodes a polypeptide having 3 different lengths with differentN-terminal sequences.

[0443] The remainder of fractions 7 to 14 was concentrated by TCAprecipitation and the precipitate was subjected to SDS-PAGE using a 10%gel under reducing conditions. The protein bands were then transferredfrom the polyacrylamide gel onto a polyvinylidine difluoride (PVDF) film(ProBlot™, Applied Biosystems) using a gel membrane transfer device(Marisol, KS-8441) operating at 9 V in the presence of transfer buffer[0.02% SDS, 20% methanol, 25 mM Tris-Borate (pH 9.5)] at 4° C. for 2.5hours.

[0444] After this time, the membrane was stained with 0.2% naphthol blueblack (Sigma), and the three bands corresponding to those previouslyidentified were excised from the membrane, and the sequence of each bandwas determined to the 6th amino acid from the N-terminal using a gasphase protein sequencer (Shimadzu, PPSQ-10). The N terminal of the bandwith the second largest apparent molecular weight (molecular weightabout 60,000) was as follows:

[0445] Val-Val-Phe-Val-Lys-Gln (amino acid nos. 1 to 6 of sequence IDno. 12)

[0446] These six amino acids correspond to the first six amino acids ofthe ORF from clone 31-7 and also correspond to the sequence of six aminoacids starting from the 24th amino acid (val) from the N terminal of theprecursor polypeptide encoded by the cDNA inserts of pSRα31-7His andpSRα31-7. Accordingly, deletion of amino acid numbers 1 to 23 from the Nterminal of this precursor polypeptide should result in the secretion ofa mature form of the protein starting with a Val residue.

EXAMPLE 14

[0447] Determination of Reducing Activity

[0448] i) Construction of an Expression Vector

[0449] The polypeptide purified in the previous Examples was onlyobtainable in extremely small amounts as it was expressed from COS-1cells. It was not, therefore, possible to use the polypeptide for otherpurposes, such as activity assays. Accordingly, it was necessary to finda way to express the polypeptide encoded by the cDNA insert of pSRα31-7in an alternative host permitting production of suitable quantities forpurification and assaying. To achieve this, the following procedure wasperformed.

[0450] pUCKM31-7 was digested with HindIII, the 3003 bp fragmentcontaining the cDNA insert was isolated and purified and the terminalswere blunted using a DNA blunting kit. The fragment was then furtherdigested with XbaI.

[0451] The expression vector pMAL-c [Guan, C. et al. (1987) Gene 67,21-30] was digested with XbaI and StuI, and then the above XbaI-modifiedHindII fragment was ligated into this cleaved plasmid using T4 DNAligase. The resulting construct is shown in FIG. 11. The construct wasthen used to transform E. coli TB-1 and Amp^(R) transformants wereselected and screened. A strain in which the direction of cDNAtranscription was identical to the direction of the promoter wasselected, and the plasmid thus obtained was designated pMAL31-7.

[0452] ii) Expression and Purification of Fusion Protein

[0453] A seed culture of E. coli harboring pMAL31-7 was prepared byculturing with shaking overnight at 37° C. in 3 ml of LB mediumcontaining 50 μg/ml of ampicillin. The following day, 1 ml of the seedculture was added to 100 ml of fresh LB culture medium containing 50μg/ml of ampicillin and cultured with shaking at 37° C. until theOD_(600 nm) reached 0.5. At this stage, IPTG was added to the culture toa final concentration of 0.1 mM, and the culture broth was furthercultured with shaking overnight at 37° C.

[0454] The following day, bacterial cells were recovered from theovernight culture by centrifuging at 6500 rpm for 20 minutes at 4° C.The pellet was then suspended in 10 ml of column buffer and the cells inthe resulting suspension were disrupted by treating with an ultrasonicdisintegrator. Whole cells and cell fragments were then removed bycentrifuging at 8800 rpm for 30 minutes at 0° C., and the solubleprotein fraction was recovered as the supernatant. 1 ml of this solublefraction was then subjected to chromatography on an amylose resin column(New England Biolabs).

[0455] The elution buffer for the chromatography was prepared by addingmaltose to 10 ml of the column buffer to a final concentration of 10 mM.

[0456] The negative control sample was also chromatographed. Thisnegative control was prepared using a similar procedure, except that thepMAL-c vector was used without any cDNA insert. The reducing activity ofthe protein in the chromatography samples was then assayed.

[0457] iii) Determination of Reducing Activity

[0458] Determination of reducing activity was performed in a cuvette(SARSTEDT, 10×4×45 mm) using dichlorophenol-indophenol (DCIP) andoxidized glutathione.

[0459] a) Determination of Reducing Activity Using DCIP

[0460] 90.4 μg, as determined using the Protein Assay Kit (Bio-Rad), ofeach of the chromatography samples obtained in ii) above were separatelymixed with 1 ml of 50 μM DCIP (Sigma). 15 μl of 1 mM NADPH(Boehringer-Mannheim) were then added to each of the samples and theOD_(600 nm) and OD_(340 nm) absorbance values were monitored with time.The resulting decrease in absorbance at both wavelengths as shown inFIG. 12, and it can be seen that only the pMAL31-7 sample contains afactor that reduces DCIP.

[0461] b) Determination of Reducing Activity Using Oxidized Glutathione

[0462] 15 ml of 10 mM oxidized glutathione (Boeringer-Mannheim) wereadded to 90.4 μg of each of the chromatography samples obtained in ii)above and which had previously been loaded into separate cuvettes. 15 μlof 1 mM NADPH were added to each cuvette, and the absorbance atOD_(340 nm) was monitored with time. The results are shown in FIG. 13,and it can be seen that only the protein from the pMAL31-7 sample iscapable of reducing oxidized glutathione. It was also observed thatthere is no consumption of NADPH when no oxidized glutathione ispresent, so that it was concluded that the protein from the pMAL31-7sample can only reduce oxidized glutathione in the presence of NADPH.

EXAMPLE 15

[0463] Purification and Analysis of N-Terminal Amino Acid Sequence

[0464] From Example 13, it was concluded that COS-1 cells transfectedwith pSRα31-7His expressed a polypeptide having three types ofN-terminal.

[0465] In a separate experiment, rabbits were immunized with fusionprotein recovered from E. coli transformed with pMAL31-7 to obtain apolyclonal antibody preparation against KM31-7 protein. Western blottingwas performed using this polyclonal antibody, and it was clear thatthree types of bands are also detected in the serum-free culturesupernatant obtained from COS-1 cells transfected with pSRα31-7. Thisresult is similar to that obtained in Example 13.

[0466] Accordingly, COS-1 cells were transfected with pSRα31-7 with theaim of collecting of a large volume of serum-free culture supernatant toallow purification and analysis of the N-terminal sequence of the KM31-7protein.

[0467] COS-1 cells were transfected with pSRα31-7 and were cultured for3 days in 150 mm petridishes each containing 30 ml of DMEM. The culturesupernatant was harvested after this time, and 30 ml of fresh mediumwere added to each dish and culture was continued for a further threedays. Once again, the culture supernatant was harvested. Other aspectsof the transfection and culture were as described in Example 11, but 199dishes were cultured.

[0468] The harvested supernatants were pooled and 10 liters ofserum-free culture supernatant were collected after centrifugation andthis was dialyzed overnight against 10 mM Tris-HCl (pH 9.0). Ionexchange chromatography was then performed eight times on the dialyzedpreparation under the following conditions using FPLC (Pharmacia):

[0469] Column: 20 ml of DEAE Sepharose Fast Flow (Pharmacia) filled intoXK16/20 (φ2.0×20 cm, Pharmacia)

[0470] Elution buffers:

[0471] A) 10 mM Tris-HCl (pH 9.0)

[0472] B) 10 mM Tris-HCl (pH 9.0)−0.5 M NaCl

[0473] Flow rate: 1 ml/min

[0474] Fraction solution: 3 ml/tube

[0475] Elution conditions: Elution buffer A changing over to Elutionbuffer B in a linear concentration gradient over a period of 60 minutes.

[0476] The fractions eluted at each NaCl concentration from 0.1 M to 0.4M were collected and pooled, and dialyzed overnight against a dialysisbuffer containing 0.1 M Tris-HCl, 5 mM EDTA (pH 7.6) and 1 mM2-mercaptoethanol. The dialyzed preparation was then subjected toaffinity chromatography using 2′,5′-ADP Sepharose 4B (Pharmacia) underthe following conditions:

[0477] Column: 20 ml of 2′,5′-ADP Sepharose 4B filled into XK16/20(φ2.0×20 cm, Pharmacia)

[0478] Elution buffers:

[0479] A) 0.1 M Tris-HCl, 5 mM EDTA (pH 7.6), 1 mM 2-mercaptoethanol

[0480] B) 0.1 M Tris-HCl, 5 mM EDTA (pH 7.6), 1 mM 2-mercaptoethanol, 10mM NADPH

[0481] Flow rate: 0.5 ml/min

[0482] Fraction solution: 2 ml/tube

[0483] Elution conditions: Elution buffer A changing over to Elutionbuffer B in a linear concentration gradient over a period of 120minutes.

[0484] 100 μl aliquots of each of the resulting fractions wereprecipitated with TCA and the precipitates were subjected to SDS-PAGEusing a 12.5% gel under reducing conditions.

[0485] After electrophoresis, the gel was silver-stained to detect theprotein bands. Three bands were obtained starting with fraction #11.

[0486] The whole of the remainder of fractions #11 to #14 were thenconcentrated by TCA precipitation and the precipitate was subjected toSDS-PAGE using a 12.5% gel under reducing conditions. The protein wasthen transferred from the gel to a ProBlot PVDF membrane (AppliedBiosystems) following electrophoresis. After transfer of the protein tothe membrane, the mebrane was stained with 0.2% naphthol blue black andthe three proteinaceous bands were excised. N terminal sequence analysiswas then performed using a gaseous phase protein sequencer.

[0487] The N terminal of the band apparently having the smallestmolecular weight of the three types was determined to beLys-Leu-Leu-Lys-Met. These five amino acids correspond to the five aminoacids starting from the 49th amino acid from the N terminal of thepolypeptide encoded by the cDNA insert of pSRα31-7. Accordingly, it wasconcluded that cleavage of the peptide at the 48th residue resulted inone mature form of the protein starting with an N-terminal Lys.

EXAMPLE 16

[0488] Preparation of a Monoclonal Antibody against the KM31-7 Protein

[0489] (a) Preparation of Antigen Protein

[0490] A seed culture of E. coli harboring pMAL31-7 was prepared byculturing a loop of cells with shaking overnight at 37° C. in 3 ml of LBmedium containing 50 μg/ml of ampicillin. 1 ml of the resulting seedculture was inoculated into 100 ml of fresh LB medium containing 50μg/ml of ampicillin and this was cultured with shaking at 37° C. untilthe OD_(600 nm) reached 0.5. At this stage, IPTG was added to theculture broth a final concentration of 0.1 mM which was then furthercultured with shaking overnight at 37° C.

[0491] Cells were recovered from the resulting overnight culture bycentrifugal separation at 6500 r.p.m. for 20 minutes at 4° C., and thepellet was suspended in 10 ml of column buffer. The cells in theresulting suspension were disrupted using an ultrasonic disintegratorand the resulting liquid was centrifuged at 8,000 r.p.m. and at 0° C.for 30 minutes. The resulting supernatant contained the soluble proteinfraction.

[0492] This soluble protein fraction was subjected to chromatography ona 1 ml amylose resin column. Elution was performed with 10 ml of columnbuffer containing 10 mM of maltose. The fusion protein obtained from thechromatography was then stored and subsequently used as the antigen.

[0493] (b) Preparation of Immunized Mice Spleen Cells

[0494] 2 ml of Freund's complete adjuvant was added to 2 ml of theantigen (equivalent to 200 μg) purified in a) above to form an emulsion.This emulsion was taken up in a 5 ml syringe barrel equipped with aglass junction, and the emulsion was used to immunize 8 week-old, maleBALB/c mice by subcutaneous injection.

[0495] Starting with the second round of immunization, Freund'sincomplete adjuvant was used as the adjuvant, but following the sameprocedure as with the first immunization. Immunization was performedfour times altogether, at a rate of one immunization roughly every 2weeks.

[0496] Starting with the second immunization, blood was sampled from thevenous plexus of the fundus oculi immediately before immunization, andthe titer of anti-KM31-7 antibody in the serum was determined bysolid-phase, enzyme-linked immunosorbent assay (ELISA).

[0497] Solid Phase Anti-KM31-7 ELISA

[0498] Between 150 and 200 μl (corresponding to about 200 ng of fusionprotein) of the serum-free culture supernatant obtained from COS-1 cellstransfected with pSRα31-7 were placed in each well of a 96 well ELISAplate (Costar) for use as the antigen. The plate was then allowed tostand overnight at 4° C. to coat the bottom surfaces of the plate wells.The following day, the plate was washed 3 times with 0.1% Tween20/phosphate buffered saline (0.1 Tween 20/PBS) and then each well wasloaded with 100 μl of BSA prepared to 10 μg/ml with PBS and allowed tostand at room temperature for 1 hour.

[0499] After this time, the plate was washed for a further three timeswith 0.1% Tween 20/PBS. 30-100 μl of primary antibody in the form of aserially diluted sample (for example, mouse serum, hybridoma culturesupernatant or monoclonal antibody) were loaded into each well, and theplate was allowed to stand at room temperature for 1 hour.

[0500] After this time, the plate was again washed three times with 0.1%Tween 20/PBS and then 100 μl of secondary antibody was added to eachwell. The secondary antibody was prepared as a 3000-fold dilutionsolution of goat anti-mouse IgG-peroxidase complex (Amersham) or a3000-fold dilution solution of goat anti-mouse IgG alkaline phosphatasecomplex (BIO-RAD). The plate was then allowed to stand at roomtemperature for 1 to 2 hours.

[0501] After this time, the plate was again washed three times with 0.1%Tween 20/PBS, and then 100 μl of either peroxidase substrate solution(BIO-RAD, Peroxidase Substrate Kit ABTS) or 10% diethanolaminecontaining 0.001% para-nitrophenyl phosphate solution were added to eachwell. The plate was then allowed to stand at room temperature for 15 to30 minutes, whereafter the antibody titer could be calculated bymeasuring the absorbance at 415 nm or 405 nm using a microplate reader(BIO-RAD).

[0502] (c) Preparation of Mouse Myeloma Cells

[0503] 8-Azaguanine-resistant mouse myeloma cells P3-X63-Ag8.653 (653)(ATCC no. CRL-1580) were cultured in a normal medium (complete GIT) toobtain a minimum of 2×10⁷ cells.

[0504] (d) Preparation of Hybridoma

[0505] 1.4×10⁸ immunized mouse spleen cells obtained after theimmunization regimen described in b) above were thoroughly washed withDMEM (Nissui Pharmaceutical). The washed cells were then mixed with1.5×10⁷ mouse myeloma cells P3-X63-Ag8.653 (653) prepared in c) above,and the resulting mixture was centrifuged at 800 r.p.m. for 6 minutes.

[0506] The cell group, consisting of a mixture of the spleen cells andP3-X63-Ag8.653 (653) cells, was collected as the pellet and was brokenup. Polyethylene glycol 4000 (polyethylene glycol #4000) as a 50%solution with DMEM was prepared in advance, and this solution wasdripped onto the broken up cells over a period of 1 minute with stirringat a rate of 2 ml/min. DMEM was then added to the cell preparation in asimilar manner for 1 minute at a rate of 2 ml/min. This procedure wasrepeated one more time for both of the polyethylene glycol and DMEMsolutions. Finally, 16 ml of DMEM was added gradually over a period of 3minutes. The resulting cell preparation was then centrifuged at 800r.p.m. for 6 minutes. The resulting supernatant was discarded and thecells were suspended in 35 ml of complete GIT containing 5 to 10 ng/mlof mouse IL-6.

[0507] (e) Screening of Hybridomas

[0508] 100 μl of the suspension prepared in d) above were loaded intoeach well of a 96 well plate (Sumitomo Bakelite) which was then culturedat 37° C. in a 7.5% CO₂ incubator. After 7 days of incubation, 50 μl ofHAT medium were added to each well. After a further 4 days ofincubation, another 50 μl of HAT medium were added to each well. Theplate was then incubated for 3 more days. After this time, a portion ofthe culture supernatant was sampled from wells in which colony growth offused cells could be observed, and the titer of anti-KM31-7 antibody wasassayed by the solid-phase ELISA described in b) above. Sampled mediumwas immediately replaced with HT medium.

[0509] (f) Cloning

[0510] Cloning of cells from wells testing as positive was repeatedthree times by limiting dilution analysis. Those clones observed to havea consistent antibody titer were selected for use as anti-KM31-7monoclonal antibody-producing hybridoma cell lines. At this stage inparticular, ELISA was performed not only as described in b) above, butalso a control ELISA was performed using the serum-free culturesupernatant obtained from COS-1 cells transfected with pcDL-pSRα296 toprepare the solid phase. Accordingly, those cell lines that reacted tothe former but did not react to the control ELISA were selected forcloning.

[0511] (g) Purification of Monoclonal Antibody

[0512] Culture supernatant from the anti-KM31-7 monoclonalantibody-producing hybridoma cell line was collected, filter sterilizedwith a 0.22 μm filter (Millipore), and then the antibody was purifiedusing MAbTrap GII (Pharmacia).

[0513] (h) Assaying the Monoclonal Antibody

[0514] 1) Antigen Specificity of the Monoclonal Antibody

[0515] Monoclonal antibodies were confirmed to be specific for KM31-7protein by the immune precipitation test using the serum-free culturesupernatant obtained from COS-1 cells transfected with pSRα31-7.

[0516] 2) Classification of the Monoclonal Antibody

[0517] This test was performed using a mouse monoclonal antibodyisotyping kit (Amersham), and the antibody was identified as belongingto the IgG1 subclass.

EXAMPLE 17

[0518] Isolation and Purification of KM31-7 Protein Using anAntigen-Antibody Reaction

[0519] This was performed as described in Example 16 h) 1) above. Thesame test was also repeated using the antibody and serum-freesupernatant obtained from COS-1 cells transfected with pcDL-pSRα296.

[0520] 1.4 μg of monoclonal antibody was added to 1.7 ml of each of theserum-free supernatants and allowed to react at room temperature for 1hour while centrifuging at 20 r.p.m. in 2.2 ml microcentrifuge tubes.The control was performed using serum-free supernatant obtained fromCOS-1 cells transfected with pSRα31-7 but without adding monoclonalantibody.

[0521] 30 μl of Protein G Sepharose 4 Fast Flow (Pharmacia), which hadpreviously been washed with 0.1% Tween 20/PBS, were added to each tubeto adsorb the antibody and centrifuging was continued at a speed of 20r.p.m. for 30 minutes at room temperature.

[0522] After this time, each mixture was centrifuged for several secondsat 10000 r.p.m. in a microcentrifuge, and then the supernatant wascarefully discarded so as not to lose any of the sediment. The pelletswere then individually washed with 0.1% Tween 20/PBS and thenmicrocentrifuged and washed in a similar manner a further 5 times.

[0523] The resulting sediment was suspended in SDS-PAGE sample buffersulution containing 10 μl of 2-mercaptoethanol. Each suspension washeated at 90° C. for 2 minutes, and then SDS-PAGE was performed underreducing conditions using a 12.5% gel. Following electrophoresis, theproduct was transferred from polyacrylamide gel to a nitrocellulose film(BIO-FAD). Western blotting was performed using the polyclonalanti-KM31-7 antibody described in Example 1, part (a) and theanti-KM31-7 monoclonal antibody was determined to specificallyprecipitate KM31-7 protein from COS-1/pSRα31-7 serum-free culturesupernatant.

EXAMPLE 18

[0524] Preparation of CYVV-NIa/KM31-7 Fusion Protein

[0525] In order to express KM31-7 protein using the CYVV-NIa proteasetechnique, it is necessary to link the 3′ terminal of the NIa gene inthe same ORF as the KM31-7 protein DNA. Accordingly, the followingtwo-stage procedure was performed.

[0526] i) Introduction of the 3′ Side Chain (SmaI-XbaI. 1006 bp) ofKM31-7 cDNA into pKSUN9

[0527] In order to obtain a SmaI-XbaI fragment (1,006 bp) containing the3′ end of KM31-7 cDNA, 7 μg of pSRα31-7 plasmid DNA were digested withthe restriction enzymes SmaI and XbaI, and the resulting fragment wascollected and purified using GENECLEAN II (Funakoshi Japan) using a 0.8%agarose gel.

[0528] Meanwhile, 5 μg of pKSUN9 plasmid DNA were similarly digestedwith SmaI and XbaI, and the cleavage fragment was dephosphorylated withbovine alkaline phosphatase (Alkaline Phosphatase E. coli C75, TakaraShuzo, Japan). The resulting dephosphorylated, linearized DNA wasligated with the SmaI-XbaI KM-31 fragment using a ligation kit (TakaraShuzo), and the resulting construct was used to transform E. coli strainJM109. Transformants were selected and screened to obtain a clonepNIa31-7SX containing a SmaI-XbaI fragment.

[0529] ii) Linking of NIa Protease and KM31-7

[0530] In order to link the C terminal sequence of NIa with theN-terminal sequence of the KM31-7 sub-type having a Val N-terminalresidue in the same reading frame, four types of polymerase chainreaction (PCR) primers were constructed, using a Perkin-Elmer JapanApplied Biosystems Model 392 DNA Synthesizer. The primers are asfollows: 5′ GGT CAG CAC AAA TTT CCA 3′ (1) 5′ AAA CAC AAC TTG GAA TGAACA ATT 3′ (2) 5′ TCA TTC CAA GTT GTG TTT GTG AAA 3′ (3) 5′ CAT AGG ATGCTC CAA CAA 3′ (4)

[0531] The first round of PCR was carried out by using 1 μg of pKSUN9plasmid DNA as the template. 100 pmol each of primers (1) and (2) and1/10 volume of 10-fold concentration Taq polymerase reaction buffersolution and finally 5 units of Taq polymerase (Takara Shuzo) were addedto the reaction solution, in that order. The PCR reaction was firstcarried out at 72° C. for 3 minutes, followed by 30 cycles of: 94° C.for 1 minute, 55° C. for 2 minutes and 72° C. for 3 minutes, andfinishing with a treatment at 72° C. for 10 minutes. Following the PCRreaction, the enhanced DNA product was subjected to 8% polyacrylamidegel electrophoresis. Strips of gel containing DNA were identified byethidium bromide staining and broken up. 300 μl of elution buffer (0.5ammonium acetate, 1 mM EDTA, pH 8.0) were added to each of the crushedstrips and incubated at 37° C. overnight. Centrifuging yielded thesupernatant containing the purified and amplified DNA.

[0532] PCR was once again performed in a similar fashion but using 1 μgof pUCKM31-7 plasmid DNA as the template and using primers (3) and (4),and the resulting DNA was purified as above.

[0533] The amplified fragment from the first PCR contained the sequenceencoding the residues from the N terminal of KM31-7 protein, startingwith Val-Val-Phe, through to 31 bp upstream from the XhoI site of NIa.The amplified fragment from the second PCR contained the sequence codingfor Asn-Cys-Ser-Phe-Gln from the C terminal of NIa through to 32 bpdownstream from the SmaI site of KM31-7 cDNA.

[0534] Accordingly, when PCR is performed using both DNA fragmentsresulting from the two PCR's along with primers (1) and (4), the resultis a hybridized strand consisting of 9 bp of the 3′ terminal of NIa and15 bp of the sequence encoding the desired N-terminal of KM31-7. Thus,it is possible to generate a fused DNA sequence with this portion as thelink.

[0535] As a result of this logic, the second round of PCR was performedin just this manner, and the enhanced fragment was collected from thegel.

[0536] iii) Introduction of NIa/KM31-7 DNA into pNIa31-7SX

[0537] 5 μg of the pNIa31-7SX plasmid DNA obtained in i) was digestedwith XhoI and SmaI, and the resulting DNA was dephosphorylated bytreatment with bovine alkaline phosphatase. The PCR product prepared inii) was also digested with XhoI and SmaI and the resulting fragment wasthen ligated with the digested, dephosphorylated pNIa31-7SX using aligation kit. The resulting construct was used to transform E. colistrain JM109.

[0538] Amp^(R) transformants were then selected and screened. Screeningwas effected by digesting with XhoI followed by electrophoresis. Cloneshaving only an 8.0 kbp band were selected. The plasmids of the selectedclones were then digested with HindIII and again electrophoresed. Theplasmid which was selected had a 330 bp band corresponding to part ofthe NIa cDNA insert. This plasmid was designated pNIa31-7V, andcontained the XhoI and SmaI PCR product.

[0539] The base sequence of clone pNIa31-7V was determined, and it wasconfirmed that the sequences encoding NIa and KM31-7 were linked in thesame ORF, with the necessary Gln-Val cleavage sequence located betweenNIa and the KM31-7 ptotein.

[0540] iv) Production of KM31-7 Protein

[0541] Western blotting confirmed that pNIa31-7V was functioning in E.coli and that the KM-31-7 protein was being expressed by the constructedrecombinant gene.

[0542] A seed culture of E. coli harboring the plasmid pNIa31-7V wascultured overnight with shaking in 3 ml of LB medium containing 50 μg/mlof ampicillin. One ml of the seed culture was added to 100 ml of freshLB medium containing 50 μg/ml of ampicillin and cultured at 37° C. withshaking until the OD_(600 nm) reached 1.0. At this stage, IPTG was addedto the culture broth to a final concentration of 1 mM, and the culturewas then incubated at 28° C. for two further nights with shaking.

[0543] After this time, 1 ml of the culture was transferred to amicrocentrifuge tube and centrifuged for 5 minutes at 15000 rpm. Thesupernatant was discarded and the pellet was mixed with 300 μl ofsterile water and 300 μl of SDS-PAGE sample buffer solution containing2-mercaptoethanol to break up the settled cell bodies. The resultingsuspension was heated at 95° C. for 2 minutes and then 10 μl of thissuspension were subjected to SDS-PAGE on an 8% gel under reducingconditions.

[0544] After electrophoresis, the protein was transferred from the gelonto a nitrocellulose membrane. This was achieved by contacting the gelwith the membrane and incubating in the presence of a transcriptionbuffer solution (25 mM Tris-HCl, 1.4% glycine and 20% methanol) at 4° C.for 2.5 hours and at 19 V using a gel membrane transcription apparatus(Marisol Japan).

[0545] The nitrocellulose membrane was then washed with 20 ml of PBS-Tmedium, and then blocking was performed for 1 hour in 20 ml of PBS-Tcontaining 5% skim milk (Snow Brand Co., Ltd). After this time, themembrane was rinsed with two lots of 20 ml of PBS-T and then allowed toreact for 90 minutes in 20 ml of PBS-T containing 1 μl of 100-folddiluted anti-KM31-7 rabbit MAb serum (primary antibody) in sterilewater. The nitrocellulose film was then rinsed once for 15 minutes andthen twice for 5 minutes each with 20 ml of PBS-T.

[0546] The washed membrane was then placed in a bath of 3,000-folddiluted peroxidase-labelled anti-rabbit IgG goat antibody (BIO-RAD) inPBS-T (used as the secondary antibody above), and allowed to stand for 1hour. The membrane was then washed with 20 ml of PBS-T and transferredinto a bath of ECL detection reagent (Amersham), and the bands thatreacted with anti-KM31-7 antibody were detected by autoradiography.

[0547] Western blotting was performed and a band having a molecularweight of roughly 60,000 was detected. This band demonstrated the samemobility as the protein having the second largest molecular weight ofthe three KM31-7 proteins detected from the serum-free culturesupernatant obtained by transfecting COS-1 cells with pSRα31-7 used asthe control.

MEDIA

[0548] x M Phosphate Buffer

[0549] An x M solution of Na₂HPO₄ adjusted to the desired pH using an xM solution of NaH₂PO₄.

[0550] Inoculation Buffer

[0551] 0.1 M Tris-HCl buffer, pH 7.0, 0.05 M EDTA, 1% 2-mercaptoethanol.

[0552] Extraction Buffer

[0553] 0.1 M Tris-HCl buffer, 0.05 M EDTA, 1% 2-mercaptoethanol, pH 7.0.

[0554] Degradation Solution

[0555] 200 mM ammonium carbonate, 2% SDS, 2 mM EDTA, 400 μg/ml bentoniteand 20 μg/ml protease K (pH 9.0).

[0556] 1×SSC

[0557] 0.15 M NaCl, 0.015 M trisodium citrate, pH 7.0.

[0558] Liquid LB Medium

[0559] 10 g of Bacto Tryptone (Difco), 5 g of Bacto yeast extract(Difco) and 5 g of sodium chloride, all made up to 1 liter withdistilled water.

[0560] Tris-Calcium Buffer

[0561] 10 mM Tris, 50 mM calcium choride, adjusted to pH 7.4 withhydrochloric acid.

[0562] Lysis Buffer

[0563] 0.17 g of sucrose, 250 μl of 1 M Tris-HCl buffer (pH 8.0), 200 μlof 0.5 M EDTA (pH 8.0), made up to 20 ml with redistilled water.

[0564] Alkaline-SDS Solution

[0565] 0.2 M sodium hydroxide, 1% SDS.

[0566] TBE Solution

[0567] 100 mM Tris, 100 mnM boric acid, 1 mM EDTA.

[0568] Denaturation Solution

[0569] 1.5 M sodium chloride, 0.5 M sodium hydroxide.

[0570] Neutralization Buffer

[0571] 0.5 M Tris, 3 M sodium chloride (pH 7.4).

[0572] 50× Denhardt's Solution

[0573] 1% polyvinyl pyrrolidone, 1% bovine serum albumin, 1% Ficoll 400.This solution is then diluted with redistilled water, as appropriate, toachieve the desired concentration

[0574] 5× Denaturation Buffer

[0575] 125 μl of 1 M glycine (pH 9.0), 25 μl of 1 M magnesium chloride,850 μl of redistilled water.

[0576] 5× Labelling Buffer

[0577] 25 μl of 1 M Tris-HCl buffer (pH 7.9), 5 μl of 1 M magnesiumchloride, 2.5 μl of 1 M dithiothreitol, 9.2 μl of redistilled water.

[0578] 10× M9 Salt Solution

[0579] 0.145 M disodium hydrogenphosphate, 0.172 M potassiumdihydrogeniphosphate, 0.187 M ammonium chloride, 0.137 M sodiumchloride, pH 7.0.

[0580] M9 Minimum Acrar Medium

[0581] 10 ml of 10× M9 salt solution, 100 μl of 1 M magnesium sulfate, 1ml of 20% glucose, 50 μl of 1% thiamine hydrochloride salt, 1 ml of 0.01M calcium chloride and 13 ml of redistilled water, all mixed, sterilizedby filtration, and then poured onto plates immediately after theaddition of 50 ml of 3% bactoagar.

[0582] Liquid SOB Medium

[0583] 10 g of bactotryptone, 2.5 g of bactoyeast extract, 100 μl of 5 Msodium chloride and 125 μl of 1 M potassium chloride are mixed and madeup to 500 ml with distilled water. After autoclaving, 5 ml of 1 Mmagnesium chloride and 5 ml of 1 M magnesium sulfate are added.

[0584] TFB1 Buffer

[0585] 5 ml of 1 M 2-(N-morpholino)ethanesulfonic acid (MES—adjusted topH 6.2 with 1 N HCl), 6.045 g of rubidium chloride, 0.735 g of calciumchloride bihydrate and 4.94 g of manganese chloride tetrahydrate mixed,adjusted to pH 5.8 with glacial acetic acid, made up to 500 ml withredistilled water and sterilized by filtration.

[0586] TFB2 Buffer

[0587] 1 ml of 1 M 2-(N-morpholino)propanesulfonic acid (MOPS), 1.102 gof calcium chloride bihydrate, 0.12 g of rubidium chloride and 15 ml ofglycerol mixed, adjusted to pH 6.5 with glacial acetic acid, made up to100 ml with redistilled water and sterilized by filtration.

[0588] Liquid SOC Medium

[0589] 5 ml of liquid SOB Medium, 90 μl of 20% glucose.

[0590] 2× YT Medium

[0591] 16 g of bactotryptone, 5 g of bactoyeast extract and 5 g ofsodium chloride mixed and made up to 1 liter with redistilled water.

[0592] PBS-Tw Medium

[0593] 80 mM disodium hydrogen-phosphate, 20 mM sodiumdihydrogenphosphate, 100 mM sodium chloride, 0.1% Tween 20.

[0594] PBS-T Medium

[0595] 4 g of sodium chloride, 0.1 g of potassium dihydrogenphosphate,1.45 g of disodium hydrogenphosphate dodecahydrate, 0.1 g of potassiumchloride and 0.1 g of sodium azide, all made up to 1 liter withredistilled water, pH 7.4.

[0596] Alkaline Phosphatase Substrate Solution

[0597] 0.01% p-nitrophenyl phosphate dissolved in 10% aqueousdiethanolamine solution which had been adjusted to pH 9.8 withhydrochloric acid.

[0598] Medium A

[0599] DMEM (Dulbecco's modified Eagle medium, containing 4.5 g/l ofglucose), 10% inactivated fetal bovine serum (manufactured by Hyclone)and 10 mM HEPES (pH 7.2).

[0600] Medium B

[0601] DMEM (containing 4.5 g/l of glucose), 10 mM HEPES (pH 7.2), 3%inactivated fetal bovine serum, 5 μg/ml bovine insulin (manufactured bySigma), 8 μg/ml d-biotin (manufactured by Sigma), 4 μg/ml pantothenicacid (manufactured by Sigma), 1.0 μM dexamethasone (manufactured bySigma) and 0.5 mM isobutylmethylxanthine (manufactured by Aldrich).

[0602] Medium C

[0603] DMEM (containing 4.5 g/l of glucose) containing 5% inactivatedfetal bovine serum, 10 mM HEPES (pH 7.2) and 100 ng/ml bovine insulin.

[0604] Medium D

[0605] DMEM (containing 4.5 g/l of glucose), 5% inactivated fetal bovineserum, 10 mM HEPES (pH 7.2), 100 ng/ml bovine insulin and 10 U/ml sodiumheparin (manufactured by Novo Industry Co.).

[0606] LPL Substrate Solution

[0607] 13 mM glycerol tri[9,10(n)-³H]oleate (51.8 KBq/μmol, manufacturedby Amersham), 1.3 mg/ml L-α-phosphatidylcholine distearoyl (manufacturedby Sigma Co.), 20 mg/ml bovine serum albumin (manufactured by SigmaCo.), 135 mM Tris hydrochloride [Tris-HCl (pH 8.1), manufactured bySigma Co.], 16.5% (v/v) glycerol and 16.5% (v/v) inactivated fetalbovine serum.

[0608] Guanidine Thiocyanate Solution

[0609] 4 M guanidine thiocyanate, 1% Sarkosyl, 20 mM ethylenediaminetetraacetic acid (EDTA), 25 mM sodium citrate (pH 7.0), 100 mM2-mercaptoethanol and 0.1% antifoam A (Sigma).

[0610] Adsorption Buffer

[0611] 0.5 M NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA and 0.1% SDS.

[0612] Eluting Solution

[0613] 10 mM Tris-HCl (pH 7.5), 1 mM EDTA and 0.05% SDS.

[0614] Reverse Transcriptase Reaction Solution of Example 2

[0615] 50 ml Tris-HCl (pH 8.3), 8 mM MgCl₂, 30 mM KCl, 0.3 mMdithiothreitol, 2 mM dATP, 2 mM dGTP, 2 mM dTTP, 10 μCi [α-³² P]dCTP and1.4 μg of vector primer-DNA (3′-oligo(dT)-tailed pcDV-1, Pharmacia).

[0616] Terminal Transferase Reaction Solution

[0617] 140 mM potassium cacodylate, 30 mM Tris-HCl (pH 6.8), 1 mM CoCl₂,0.5 mM dithiothreitol, 0.2 μg of poly A and 100 mM dCTP.

[0618] Restriction Enzyme Buffer

[0619] 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl₂ and 1 mMdithiothreitol.

[0620] 10-Volume Ligase Buffer

[0621] 10 mM ATP, 660 mM Tris-HCl (pH 7.5), 66 mM MgCl₂ and 100 mMdithiothreitol.

[0622] Electrophoresis Pigment

[0623] 50% glycerol, 0.01 M disodium hydrogen phosphate (pH 7.0) and0.4% bromophenol blue.

[0624] 1× TAE

[0625] 0.04 M Tris-acetate, 0.001 M EDTA.

[0626] 1×SSCP

[0627] 120 mM NaCl, 15 mM sodium citrate, 13 mM potassium dihydrogenphosphate and 1 mM EDTA.

[0628] Reverse Transcriptase Reaction Solution of Example 6

[0629] 1× first strand synthesis buffer, 5% sodium pyrophosphate, 100units of ribonuclease inhibitor, 1 mM dATP, 1 mM dGTP, 1 mM dTTP, 0.5 mMdCTP and 3.75 μg of oligo(dT) primer, all provided with the cDNA CloningSystem (Amersham).

[0630] SM Buffer

[0631] 100 mM NaCl, 8 mM MgSO₄.7H₂O, 50 mM Tris-HCl (pH 7.5) and 0.01%gelatin.

[0632] Dialysis Buffer

[0633] 20 mM phosphate buffer (pH 7.8) and 0.5 M NaCl.

[0634] Column Buffer

[0635] 10 mM Tris-HCl (pH 7.4), 200 mM NaCl and 1 mM EDTA.

[0636] Taq Polymerase Reaction Buffer Solution

[0637] Taq containing 500 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl₂,100 mM DATP, 100 mM dCTP, 100-mM dGTP, 100 mM dTTP and 2 mg/ml ofgelatin.

1 19 1320 base pairs nucleic acid double linear cDNA to mRNA N N CloverYellow Vein Virus CDS 1..1320 mat_peptide 10..1311 1 AAG TTC CAA GGG AAAAGT AAG AGA ACA AGA CAA AAG TTG AAG TTC AGA 48 Lys Phe Gln Gly Lys SerLys Arg Thr Arg Gln Lys Leu Lys Phe Arg 1 5 10 15 GCG GCA AGA GAC ATGAAG GAT CGT TAT GAA GTG CAT GCC GAT GAG GGG 96 Ala Ala Arg Asp Met LysAsp Arg Tyr Glu Val His Ala Asp Glu Gly 20 25 30 ACT TTA GTG GAA AAT TTTGGA ACT CGT TAT TCA AAG AAA GGC AAG ACA 144 Thr Leu Val Glu Asn Phe GlyThr Arg Tyr Ser Lys Lys Gly Lys Thr 35 40 45 AAA GGT ACT GTT GTG GGT TTGGGT GCA AAA ACA AGA CGG TTC ACT AAC 192 Lys Gly Thr Val Val Gly Leu GlyAla Lys Thr Arg Arg Phe Thr Asn 50 55 60 ATG TAT GGT TTT GAC CCC ACG GAGTAT TCA TTT GCT AGG TAT CTT GAT 240 Met Tyr Gly Phe Asp Pro Thr Glu TyrSer Phe Ala Arg Tyr Leu Asp 65 70 75 80 CCA ATC ACG GGT GCA ACA TTG GATGAA ACC CCA ATT CAC AAT GTA AAT 288 Pro Ile Thr Gly Ala Thr Leu Asp GluThr Pro Ile His Asn Val Asn 85 90 95 TTG GTT GCT GAG CAT TTT GGC GAC ATAAGG CTT GAT ATG GTT GAC AAG 336 Leu Val Ala Glu His Phe Gly Asp Ile ArgLeu Asp Met Val Asp Lys 100 105 110 GAG TTA CTT GAC AAA CAG CAC TTA TACCTC AAG AGA CCA ATA GAA TGT 384 Glu Leu Leu Asp Lys Gln His Leu Tyr LeuLys Arg Pro Ile Glu Cys 115 120 125 TAC TTT GTA AAG GAT GCT GGT CAG AAGGTG ATG AGG ATT GAT CTA ACA 432 Tyr Phe Val Lys Asp Ala Gly Gln Lys ValMet Arg Ile Asp Leu Thr 130 135 140 CCC CAC AAC CCA TTG TTG GCA AGC GATGTT AGC ACA ACC ATA ATG GGT 480 Pro His Asn Pro Leu Leu Ala Ser Asp ValSer Thr Thr Ile Met Gly 145 150 155 160 TAT CCT GAG AGA GAA GGT GAA CTCCGT CAA ACT GGA AAG GCA AGG TTA 528 Tyr Pro Glu Arg Glu Gly Glu Leu ArgGln Thr Gly Lys Ala Arg Leu 165 170 175 GTC GAC CCA TCA GAG TTG CCC GCGCGG AAT GAG GAT ATT GAT GCA GAG 576 Val Asp Pro Ser Glu Leu Pro Ala ArgAsn Glu Asp Ile Asp Ala Glu 180 185 190 TTT GAG AGT CTA AAT CGC ATA AGTGGT TTG CGC GAC TAT AAT CCC ATT 624 Phe Glu Ser Leu Asn Arg Ile Ser GlyLeu Arg Asp Tyr Asn Pro Ile 195 200 205 TCA CAA AAT GTT TGC TTG CTA ACAAAT GAG TCA GAA GGC CAT AGA GAG 672 Ser Gln Asn Val Cys Leu Leu Thr AsnGlu Ser Glu Gly His Arg Glu 210 215 220 AAG ATG TTT GGA ATT GGA TAT GGTTCA GTG ATC ATT ACA AAT CAA CAT 720 Lys Met Phe Gly Ile Gly Tyr Gly SerVal Ile Ile Thr Asn Gln His 225 230 235 240 CTG TTC AGA AGG AAT AAT GGGGAG TTA TCA ATT CAA TCC AAG CAT GGC 768 Leu Phe Arg Arg Asn Asn Gly GluLeu Ser Ile Gln Ser Lys His Gly 245 250 255 TAC TTC AGA TGC CGC AAC ACCACA AGC TTG AAG ATG CTG CCT TTG GAG 816 Tyr Phe Arg Cys Arg Asn Thr ThrSer Leu Lys Met Leu Pro Leu Glu 260 265 270 GGA CAT GAC ATT TTG TTG ATTCAG TTA CCA AGG GAC TTT CCA GTG TTT 864 Gly His Asp Ile Leu Leu Ile GlnLeu Pro Arg Asp Phe Pro Val Phe 275 280 285 CCA CAA AAG ATT CGC TTT AGGGAG CCA AGA GTG GAT GAC AAA ATT GTT 912 Pro Gln Lys Ile Arg Phe Arg GluPro Arg Val Asp Asp Lys Ile Val 290 295 300 TTG GTC AGC ACA AAT TTC CAGGAA AAG AGT TCC TCG AGC ACG GTC TCA 960 Leu Val Ser Thr Asn Phe Gln GluLys Ser Ser Ser Ser Thr Val Ser 305 310 315 320 GAG TCC AGT AAC ATT TCAAGA GTG CAG TCA GCC AAT TTC TAC AAG CAT 1008 Glu Ser Ser Asn Ile Ser ArgVal Gln Ser Ala Asn Phe Tyr Lys His 325 330 335 TGG ATC TCA ACA GTA GCAGGA CAC TGT GGA AAC CCT ATG GTT TCG ACT 1056 Trp Ile Ser Thr Val Ala GlyHis Cys Gly Asn Pro Met Val Ser Thr 340 345 350 AAA GAT GGA TTT ATT GTAGGT ATC CAC AGT CTT GCT TCA TTG ACA GGC 1104 Lys Asp Gly Phe Ile Val GlyIle His Ser Leu Ala Ser Leu Thr Gly 355 360 365 GAC GTT AAC ATC TTC ACAAGC TTT CCG CCG CAG TTT GAG AAC AAA TAT 1152 Asp Val Asn Ile Phe Thr SerPhe Pro Pro Gln Phe Glu Asn Lys Tyr 370 375 380 CTA CAG AAG CTC AGT GAACAC ACA TGG TGT AGT GGA TGG AAA CTA AAT 1200 Leu Gln Lys Leu Ser Glu HisThr Trp Cys Ser Gly Trp Lys Leu Asn 385 390 395 400 CTT GGA AAG ATT AGTTGG GGT GGA ATC AAC ATT GTG GAG GAT GCA CCT 1248 Leu Gly Lys Ile Ser TrpGly Gly Ile Asn Ile Val Glu Asp Ala Pro 405 410 415 GAA GAG CCC TTT ATAACA TCC AAG ATG GCA AGC CTT CTT AGT GAT TTG 1296 Glu Glu Pro Phe Ile ThrSer Lys Met Ala Ser Leu Leu Ser Asp Leu 420 425 430 AAT TGT TCA TTC CAAGCA AGT GCG 1320 Asn Cys Ser Phe Gln Ala Ser Ala 435 440 440 amino acidsamino acid linear protein Clover Yellow Vein Virus mat_peptide 4..437 2Lys Phe Gln Gly Lys Ser Lys Arg Thr Arg Gln Lys Leu Lys Phe Arg 1 5 1015 Ala Ala Arg Asp Met Lys Asp Arg Tyr Glu Val His Ala Asp Glu Gly 20 2530 Thr Leu Val Glu Asn Phe Gly Thr Arg Tyr Ser Lys Lys Gly Lys Thr 35 4045 Lys Gly Thr Val Val Gly Leu Gly Ala Lys Thr Arg Arg Phe Thr Asn 50 5560 Met Tyr Gly Phe Asp Pro Thr Glu Tyr Ser Phe Ala Arg Tyr Leu Asp 65 7075 80 Pro Ile Thr Gly Ala Thr Leu Asp Glu Thr Pro Ile His Asn Val Asn 8590 95 Leu Val Ala Glu His Phe Gly Asp Ile Arg Leu Asp Met Val Asp Lys100 105 110 Glu Leu Leu Asp Lys Gln His Leu Tyr Leu Lys Arg Pro Ile GluCys 115 120 125 Tyr Phe Val Lys Asp Ala Gly Gln Lys Val Met Arg Ile AspLeu Thr 130 135 140 Pro His Asn Pro Leu Leu Ala Ser Asp Val Ser Thr ThrIle Met Gly 145 150 155 160 Tyr Pro Glu Arg Glu Gly Glu Leu Arg Gln ThrGly Lys Ala Arg Leu 165 170 175 Val Asp Pro Ser Glu Leu Pro Ala Arg AsnGlu Asp Ile Asp Ala Glu 180 185 190 Phe Glu Ser Leu Asn Arg Ile Ser GlyLeu Arg Asp Tyr Asn Pro Ile 195 200 205 Ser Gln Asn Val Cys Leu Leu ThrAsn Glu Ser Glu Gly His Arg Glu 210 215 220 Lys Met Phe Gly Ile Gly TyrGly Ser Val Ile Ile Thr Asn Gln His 225 230 235 240 Leu Phe Arg Arg AsnAsn Gly Glu Leu Ser Ile Gln Ser Lys His Gly 245 250 255 Tyr Phe Arg CysArg Asn Thr Thr Ser Leu Lys Met Leu Pro Leu Glu 260 265 270 Gly His AspIle Leu Leu Ile Gln Leu Pro Arg Asp Phe Pro Val Phe 275 280 285 Pro GlnLys Ile Arg Phe Arg Glu Pro Arg Val Asp Asp Lys Ile Val 290 295 300 LeuVal Ser Thr Asn Phe Gln Glu Lys Ser Ser Ser Ser Thr Val Ser 305 310 315320 Glu Ser Ser Asn Ile Ser Arg Val Gln Ser Ala Asn Phe Tyr Lys His 325330 335 Trp Ile Ser Thr Val Ala Gly His Cys Gly Asn Pro Met Val Ser Thr340 345 350 Lys Asp Gly Phe Ile Val Gly Ile His Ser Leu Ala Ser Leu ThrGly 355 360 365 Asp Val Asn Ile Phe Thr Ser Phe Pro Pro Gln Phe Glu AsnLys Tyr 370 375 380 Leu Gln Lys Leu Ser Glu His Thr Trp Cys Ser Gly TrpLys Leu Asn 385 390 395 400 Leu Gly Lys Ile Ser Trp Gly Gly Ile Asn IleVal Glu Asp Ala Pro 405 410 415 Glu Glu Pro Phe Ile Thr Ser Lys Met AlaSer Leu Leu Ser Asp Leu 420 425 430 Asn Cys Ser Phe Gln Ala Ser Ala 435440 25 base pairs nucleic acid single linear other nucleic acid,synthetic DNA N N 3 GTCCATGGGG AAAAGTAAGA GAACA 25 20 base pairs nucleicacid single linear other nucleic acid, synthetic DNA N N 4 ACTCTGAGACCGTGCTCGAG 20 20 base pairs nucleic acid single linear other nucleicacid, synthetic DNA N N 5 AGGAAAAGAG TTCCTCGAGC 20 36 base pairs nucleicacid single linear other nucleic acid, synthetic DNA N N 6 AATTGTTCATTCCAAGCACC TGGGCCACCA CCTGGC 36 36 base pairs nucleic acid single linearother nucleic acid, synthetic DNA N N 7 GCCAGGTGGT GGCCCAGGTG CTTGGAATGAACAATT 36 30 base pairs nucleic acid single linear other nucleic acid,synthetic DNA N N 8 TTGTCAGCAC ACCTGGGAGC TGTAGAGCTC 30 7 amino acidsamino acid single linear protein N 9 Ala Pro Gly Pro Pro Pro Gly 1 5 7amino acids amino acid single linear protein N 10 Pro Gly Pro Pro ProGly Pro 1 5 1650 base pairs nucleic acid double linear cDNA to mRNA N NHomo sapiens KM-102 KM31-7 CDS 1..1647 mat_peptide 70..1647 sig_peptide1..69 11 ATG TCA TGT GAG GAC GGT CGG GCC CTG GAA GGA ACG CTC TCG GAA TTG48 Met Ser Cys Glu Asp Gly Arg Ala Leu Glu Gly Thr Leu Ser Glu Leu -23-20 -15 -10 GCC GCG GAA ACC GAT CTG CCC GTT GTG TTT GTG AAA CAG AGA AAGATA 96 Ala Ala Glu Thr Asp Leu Pro Val Val Phe Val Lys Gln Arg Lys Ile-5 1 5 GGC GGC CAT GGT CCA ACC TTG AAG GCT TAT CAG GAG GGC AGA CTT CAA144 Gly Gly His Gly Pro Thr Leu Lys Ala Tyr Gln Glu Gly Arg Leu Gln 1015 20 25 AAG CTA CTA AAA ATG AAC GGC CCT GAA GAT CTT CCC AAG TCC TAT GAC192 Lys Leu Leu Lys Met Asn Gly Pro Glu Asp Leu Pro Lys Ser Tyr Asp 3035 40 TAT GAC CTT ATC ATC ATT GGA GGT GGC TCA GGA GGT CTG GCA GCT GCT240 Tyr Asp Leu Ile Ile Ile Gly Gly Gly Ser Gly Gly Leu Ala Ala Ala 4550 55 AAG GAG GCA GCC CAA TAT GGC AAG AAG GTG ATG GTC CTG GAC TTT GTC288 Lys Glu Ala Ala Gln Tyr Gly Lys Lys Val Met Val Leu Asp Phe Val 6065 70 ACT CCC ACC CCT CTT GGA ACT AGA TGG GGT CTT GGA GGA ACA TGT GTG336 Thr Pro Thr Pro Leu Gly Thr Arg Trp Gly Leu Gly Gly Thr Cys Val 7580 85 AAT GTG GGT TGC ATA CCT AAA AAA CTG ATG CAT CAA GCA GCT TTG TTA384 Asn Val Gly Cys Ile Pro Lys Lys Leu Met His Gln Ala Ala Leu Leu 9095 100 105 GGA CAA GCC CTG CAA GAC TCT CGA AAT TAT GGA TGG AAA GTC GAGGAG 432 Gly Gln Ala Leu Gln Asp Ser Arg Asn Tyr Gly Trp Lys Val Glu Glu110 115 120 ACA GTT AAG CAT GAT TGG GAC AGA ATG ATA GAA GCT GTA CAG AATCAC 480 Thr Val Lys His Asp Trp Asp Arg Met Ile Glu Ala Val Gln Asn His125 130 135 ATT GGC TCT TTG AAT TGG GGC TAC CGA GTA GCT CTG CGG GAG AAAAAA 528 Ile Gly Ser Leu Asn Trp Gly Tyr Arg Val Ala Leu Arg Glu Lys Lys140 145 150 GTC GTC TAT GAG AAT GCT TAT GGG CAA TTT ATT GGT CCT CAC AGGATT 576 Val Val Tyr Glu Asn Ala Tyr Gly Gln Phe Ile Gly Pro His Arg Ile155 160 165 AAG GCA ACA AAT AAT AAA GGC AAA GAA AAA ATT TAT TCA GCA GAGAGA 624 Lys Ala Thr Asn Asn Lys Gly Lys Glu Lys Ile Tyr Ser Ala Glu Arg170 175 180 185 TTT CTC ATT GCC ACT GGT GAA AGA CCA CGT TAC TTG GGC ATCCCT GGT 672 Phe Leu Ile Ala Thr Gly Glu Arg Pro Arg Tyr Leu Gly Ile ProGly 190 195 200 GAC AAA GAA TAC TGC ATC AGC AGT GAT GAT CTT TTC TCC TTGCCT TAC 720 Asp Lys Glu Tyr Cys Ile Ser Ser Asp Asp Leu Phe Ser Leu ProTyr 205 210 215 TGC CCG GGT AAG ACC CTG GTT GTT GGA GCA TCC TAT GTC GCTTTG GAG 768 Cys Pro Gly Lys Thr Leu Val Val Gly Ala Ser Tyr Val Ala LeuGlu 220 225 230 TGC GCT GGA TTT CTT GCT GGT ATT GGT TTA GAC GTC ACT GTTATG GTT 816 Cys Ala Gly Phe Leu Ala Gly Ile Gly Leu Asp Val Thr Val MetVal 235 240 245 AGG TCC ATT CTT CTT AGA GGA TTT GAC CAG GAC ATG GCC AACAAA ATT 864 Arg Ser Ile Leu Leu Arg Gly Phe Asp Gln Asp Met Ala Asn LysIle 250 255 260 265 GGT GAA CAC ATG GAA GAA CAT GGC ATC AAG TTT ATA AGACAG TTC GTA 912 Gly Glu His Met Glu Glu His Gly Ile Lys Phe Ile Arg GlnPhe Val 270 275 280 CCA ATT AAA GTT GAA CAA ATT GAA GCA GGG ACA CCA GGCCGA CTC AGA 960 Pro Ile Lys Val Glu Gln Ile Glu Ala Gly Thr Pro Gly ArgLeu Arg 285 290 295 GTA GTA GCT CAG TCC ACC AAT AGT GAG GAA ATC ATT GAAGGA GAA TAT 1008 Val Val Ala Gln Ser Thr Asn Ser Glu Glu Ile Ile Glu GlyGlu Tyr 300 305 310 AAT ACG GTG ATG CTG GCA ATA GGA AGA GAT GCT TGC ACAAGA AAA ATT 1056 Asn Thr Val Met Leu Ala Ile Gly Arg Asp Ala Cys Thr ArgLys Ile 315 320 325 GGC TTA GAA ACC GTA GGG GTG AAG ATA AAT GAA AAG ACTGGA AAA ATA 1104 Gly Leu Glu Thr Val Gly Val Lys Ile Asn Glu Lys Thr GlyLys Ile 330 335 340 345 CCT GTC ACA GAT GAA GAA CAG ACC AAT GTG CCT TACATC TAT GCC ATT 1152 Pro Val Thr Asp Glu Glu Gln Thr Asn Val Pro Tyr IleTyr Ala Ile 350 355 360 GGC GAT ATA TTG GAG GAT AAG GTG GAG CTC ACC CCAGTT GCA ATC CAG 1200 Gly Asp Ile Leu Glu Asp Lys Val Glu Leu Thr Pro ValAla Ile Gln 365 370 375 GCA GGA AGA TTG CTG GCT CAG AGG CTC TAT GCA GGTTCC ACT GTC AAG 1248 Ala Gly Arg Leu Leu Ala Gln Arg Leu Tyr Ala Gly SerThr Val Lys 380 385 390 TGT GAC TAT GAA AAT GTT CCA ACC ACT GTA TTT ACTCCT TTG GAA TAT 1296 Cys Asp Tyr Glu Asn Val Pro Thr Thr Val Phe Thr ProLeu Glu Tyr 395 400 405 GGT GCT TGT GGC CTT TCT GAG GAG AAA GCT GTG GAGAAG TTT GGG GAA 1344 Gly Ala Cys Gly Leu Ser Glu Glu Lys Ala Val Glu LysPhe Gly Glu 410 415 420 425 GAA AAT ATT GAG GTT TAC CAT AGT TAC TTT TGGCCA TTG GAA TGG ACG 1392 Glu Asn Ile Glu Val Tyr His Ser Tyr Phe Trp ProLeu Glu Trp Thr 430 435 440 ATT CCG TCA AGA GAT AAC AAC AAA TGT TAT GCAAAA ATA ATC TGT AAT 1440 Ile Pro Ser Arg Asp Asn Asn Lys Cys Tyr Ala LysIle Ile Cys Asn 445 450 455 ACT AAA GAC AAT GAA CGT GTT GTG GGC TTT CACGTA CTG GGT CCA AAT 1488 Thr Lys Asp Asn Glu Arg Val Val Gly Phe His ValLeu Gly Pro Asn 460 465 470 GCT GGA GAA GTT ACA CAA GGC TTT GCA GCT GCGCTC AAA TGT GGA CTG 1536 Ala Gly Glu Val Thr Gln Gly Phe Ala Ala Ala LeuLys Cys Gly Leu 475 480 485 ACC AAA AAG CAG CTG GAC AGC ACA ATT GGA ATCCAC CCT GTC TGT GCA 1584 Thr Lys Lys Gln Leu Asp Ser Thr Ile Gly Ile HisPro Val Cys Ala 490 495 500 505 GAG GTA TTC ACA ACA TTG TCT GTG ACC AAGCGC TCT GGG GCA AGC ATC 1632 Glu Val Phe Thr Thr Leu Ser Val Thr Lys ArgSer Gly Ala Ser Ile 510 515 520 CTC CAG GCT GGC TGC TGA 1650 Leu Gln AlaGly Cys 525 549 amino acids amino acid linear protein 12 Met Ser Cys GluAsp Gly Arg Ala Leu Glu Gly Thr Leu Ser Glu Leu -23 -20 -15 -10 Ala AlaGlu Thr Asp Leu Pro Val Val Phe Val Lys Gln Arg Lys Ile -5 1 5 Gly GlyHis Gly Pro Thr Leu Lys Ala Tyr Gln Glu Gly Arg Leu Gln 10 15 20 25 LysLeu Leu Lys Met Asn Gly Pro Glu Asp Leu Pro Lys Ser Tyr Asp 30 35 40 TyrAsp Leu Ile Ile Ile Gly Gly Gly Ser Gly Gly Leu Ala Ala Ala 45 50 55 LysGlu Ala Ala Gln Tyr Gly Lys Lys Val Met Val Leu Asp Phe Val 60 65 70 ThrPro Thr Pro Leu Gly Thr Arg Trp Gly Leu Gly Gly Thr Cys Val 75 80 85 AsnVal Gly Cys Ile Pro Lys Lys Leu Met His Gln Ala Ala Leu Leu 90 95 100105 Gly Gln Ala Leu Gln Asp Ser Arg Asn Tyr Gly Trp Lys Val Glu Glu 110115 120 Thr Val Lys His Asp Trp Asp Arg Met Ile Glu Ala Val Gln Asn His125 130 135 Ile Gly Ser Leu Asn Trp Gly Tyr Arg Val Ala Leu Arg Glu LysLys 140 145 150 Val Val Tyr Glu Asn Ala Tyr Gly Gln Phe Ile Gly Pro HisArg Ile 155 160 165 Lys Ala Thr Asn Asn Lys Gly Lys Glu Lys Ile Tyr SerAla Glu Arg 170 175 180 185 Phe Leu Ile Ala Thr Gly Glu Arg Pro Arg TyrLeu Gly Ile Pro Gly 190 195 200 Asp Lys Glu Tyr Cys Ile Ser Ser Asp AspLeu Phe Ser Leu Pro Tyr 205 210 215 Cys Pro Gly Lys Thr Leu Val Val GlyAla Ser Tyr Val Ala Leu Glu 220 225 230 Cys Ala Gly Phe Leu Ala Gly IleGly Leu Asp Val Thr Val Met Val 235 240 245 Arg Ser Ile Leu Leu Arg GlyPhe Asp Gln Asp Met Ala Asn Lys Ile 250 255 260 265 Gly Glu His Met GluGlu His Gly Ile Lys Phe Ile Arg Gln Phe Val 270 275 280 Pro Ile Lys ValGlu Gln Ile Glu Ala Gly Thr Pro Gly Arg Leu Arg 285 290 295 Val Val AlaGln Ser Thr Asn Ser Glu Glu Ile Ile Glu Gly Glu Tyr 300 305 310 Asn ThrVal Met Leu Ala Ile Gly Arg Asp Ala Cys Thr Arg Lys Ile 315 320 325 GlyLeu Glu Thr Val Gly Val Lys Ile Asn Glu Lys Thr Gly Lys Ile 330 335 340345 Pro Val Thr Asp Glu Glu Gln Thr Asn Val Pro Tyr Ile Tyr Ala Ile 350355 360 Gly Asp Ile Leu Glu Asp Lys Val Glu Leu Thr Pro Val Ala Ile Gln365 370 375 Ala Gly Arg Leu Leu Ala Gln Arg Leu Tyr Ala Gly Ser Thr ValLys 380 385 390 Cys Asp Tyr Glu Asn Val Pro Thr Thr Val Phe Thr Pro LeuGlu Tyr 395 400 405 Gly Ala Cys Gly Leu Ser Glu Glu Lys Ala Val Glu LysPhe Gly Glu 410 415 420 425 Glu Asn Ile Glu Val Tyr His Ser Tyr Phe TrpPro Leu Glu Trp Thr 430 435 440 Ile Pro Ser Arg Asp Asn Asn Lys Cys TyrAla Lys Ile Ile Cys Asn 445 450 455 Thr Lys Asp Asn Glu Arg Val Val GlyPhe His Val Leu Gly Pro Asn 460 465 470 Ala Gly Glu Val Thr Gln Gly PheAla Ala Ala Leu Lys Cys Gly Leu 475 480 485 Thr Lys Lys Gln Leu Asp SerThr Ile Gly Ile His Pro Val Cys Ala 490 495 500 505 Glu Val Phe Thr ThrLeu Ser Val Thr Lys Arg Ser Gly Ala Ser Ile 510 515 520 Leu Gln Ala GlyCys 525 15 base pairs nucleic acid single linear other nucleic acid,synthetic DNA N N 13 TAAATAAATA AATAA 15 66 base pairs nucleic aciddouble linear other nucleic acid, synthetic DNA N N 14 CTAGCGCTCTGGGGCAAGCA TCCTCCAGGC TGGCTGCCAC CACCACCACC ACCACTGATC 60 TAGACT 66 18base pairs nucleic acid single linear other nucleic acid, synthetic DNAN N 15 GGTCAGCACA AATTTCCA 18 24 base pairs nucleic acid single linearother nucleic acid, synthetic DNA N N 16 AAACACAACT TGGAATGAAC AATT 2424 base pairs nucleic acid single linear other nucleic acid, syntheticDNA N N 17 TCATTCCAAG TTGTGTTTGT GAAA 24 18 base pairs nucleic acidsingle linear other nucleic acid, synthetic DNA N N 18 CATAGGATGCTCCAACAA 18 6 amino acids amino acid single linear protein 19 Asn CysSer Phe Gln Xaa 1 5

What is claimed is:
 1. A polynucleotide sequence encoding a fusionprotein and comprising, in the 5′ to 3′ direction and in the same openreading frame: a) a sequence encoding the clover yellow vein virusNuclear Inclusion a protein, or a mutant or variant thereof havingsimilar proteolytic specificity to that of clover yellow vein virusNuclear Inclusion a protein; b) a sequence encoding a cleavage peptiderecognizable by and cleavable by said clover yellow vein virus NuclearInclusion a protein, or said mutant or variant thereof; and c) at leastone sequence encoding a polypeptide.
 2. The polynucleotide sequence ofclaim 1, which is DNA.
 3. The polynucleotide sequence of claim 2, whichis in the form of a double strand.
 4. An antisense polynucleotidesequence corresponding to the polynucleotide sequence of claim
 1. 5. Thepolynucleotide sequence of claim 1, in which the sequence of c) encodesmore than one polypeptide and there are encoded further cleavagesequences between each encoded polypeptide.
 6. The polynucleotidesequence of claim 5, in which said further cleavage sequences arerecognizable by clover yellow vein virus Nuclear Inclusion a.
 7. Thepolynucleotide sequence of claim 1, wherein the sequence of c) encodesonly one polypeptide.
 8. The polynucleotide sequence of claim 1, inwhich the sequence of b) is wholly or partly comprised in either or bothof the sequences a) and c).
 9. The polynucleotide sequence of claim 8,in which the sequence of b) is wholly comprised in the sequences of a)and b).
 10. The polynucleotide sequence of claim 1, wherein there areencoded one or more amino acid residues between the cleavage peptideencoded by the sequence of b) and the polypeptide encoded by thesequence of c).
 11. The polynucleotide sequence of claim 1, in whichcleavage of the fusion protein encoded thereby by the Nuclear Inclusiona protein encoded by the sequence of a) yields a polypeptide having anadditional N-terminal amino acid sequence of which the terminal residueis a residue of glycine, serine or alanine.
 12. The polynucleotidesequence of claim 1, in which cleavage of a fusion protein encodedthereby by the Nuclear Inclusion a protein encoded by the sequence of a)yields a polypeptide having an additional N-terminal amino acid sequenceof which the terminal residue is a residue of glycine, serine oralanine, said residue being removable by the action of a suitableaminopeptidase if desired.
 13. The polynucleotide sequence of claim 1,in which a proline residue is encoded between the N-terminal of thepolypeptide and the C-terminal of the cleavage peptide.
 14. Thepolynucleotide sequence of claim 1, in which a proline residue isencoded between the N-terminal of the polypeptide and the C-terminal ofthe cleavage peptide, thereby permitting the cleavage of any residuesextending beyond the proline residue by the action of aminopeptidase P(3.4.11.9).
 15. The polynucleotide sequence of claim 14, in which, aftercleavage up to the the proline residue, the proline residue can then beremoved by the action of proline iminopeptidase (3.4.11.5).
 16. Thepolynucleotide sequence of claim 1, in which an alanine residue isencoded between the N-terminal of the polypeptide and the C-terminal ofthe cleavage peptide.
 17. The polynucleotide sequence of claim 1, inwhich an alanine residue is encoded between the N-terminal of thepolypeptide and the C-terminal of the cleavage peptide, therebypermitting the cleavage of any residues extending beyond the alanineresidue by the action of aminopeptidase P (3.4.11.9), and cleavage ofthe alanine residue by the catalytic action of alanine aminopeptidase(3.4.11.14).
 18. The polynucleotide sequence of claim 1, wherein thesequence of a) is given by nucleotide numbers 10 to 1311 in sequence IDnumber 1 in the sequence listing.
 19. The polynucleotide sequence ofclaim 1, wherein the sequence of a) encodes a polypeptide given by aminoacid numbers 4 to 437 in sequence ID number 2 in the sequence listing.20. A polynucleotide sequence encoding the clover yellow vein virusNuclear Inclusion a protein as given by nucleotide numbers 10 to 1311 insequence ID number 1 in the sequence listing, or a mutant or variantthereof having similar proteolytic specificity to that of clover yellowvein virus Nuclear Inclusion a protein.
 21. A vector containing thepolynucleotide sequence of claim
 1. 22. The vector of claim 21, which isan expression vector.
 23. A host transformed with the vector of claim21.
 24. A host transformed with the vector of claim
 22. 25. Anexpression system comprising a host transformed with the vector of claim22.
 26. A polypeptide expressed by the system of claim
 25. 27. A systemfor the preparation of a polypeptide, wherein a precursor form of thepolypeptide is cleaved by clover yellow virus Nuclear Inclusion aprotein, or a mutant or variant thereof.
 28. The system of claim 27,wherein said precursor is a fusion protein.
 29. A system for thepreparation of a polypeptide, wherein a precursor form of thepolypeptide containing the sequence AsnCysSerPheGlnX is cleaved byclover yellow virus Nuclear Inclusion a protein, or a mutant or variantthereof.
 30. A system for the preparation of a polypeptide, wherein aprecursor form of the polypeptide containing the sequenceAsnCysSerPheGlnX is cleaved by clover yellow virus Nuclear Inclusion aprotein, or a mutant or variant thereof, to yield the mature form of thepolypeptide.
 31. The polynucleotide sequence of claim 1, which shares atleast 90% sequence homology with residues 10 to 1311 in sequence IDnumber
 1. 32. The polynucleotide sequence of claim 1, further encoding aleader sequence upstream of the sequence a).
 33. A fusion proteinencoded by a polynucleotide sequence comprising, in the 5′ to 3′direction and in the same open reading frame: a) a sequence encoding theclover yellow vein virus Nuclear Inclusion a protein, or a mutant orvariant thereof having similar proteolytic specificity to that of cloveryellow vein virus Nuclear Inclusion a protein; b) a sequence encoding acleavage peptide recognizable by and cleavable by said clover yellowvein virus Nuclear Inclusion a protein, or said mutant or variantthereof; and c) at least one sequence encoding a polypeptide.
 34. Thefusion protein of claim 33 comprising more than one polypeptide andwherein there are located cleavage peptides between each polypeptide.35. The fusion protein of claim 34, in which said further cleavagesequences are recognizable by clover yellow vein virus Nuclear Inclusiona.
 36. The fusion protein of claim 33, which consists of only onepolypeptide in addition to the clover yellow vein virus NuclearInclusion a protein, or the mutant or variant thereof.
 37. The fusionprotein of claim 33, wherein the cleavage peptide is wholly or partlycomprised in either or both of the peptides as encoded by sequences a)and c).
 38. The fusion protein of claim 33, wherein the cleavage peptideis wholly comprised in the peptides as encoded by sequences a) and c).39. The polynucleotide sequence of claim 33, wherein one or more aminoacid residues are located between the cleavage peptide and thepolypeptide.
 40. The fusion protein of claim 33, wherein autolysis bythe peptide encoded by sequence a) yields a polypeptide having anadditional N-terminal amino acid sequence of which the terminal residueis a residue of glycine, serine or alanine.
 41. The expression system ofclaim 25, wherein autolysis by the peptide encoded by sequence a) yieldsa polypeptide having an additional N-terminal amino acid sequence ofwhich the terminal residue is a residue of glycine, serine or alanine,and an aminopeptidase in the host serves to remove said N-terminalresidue.
 42. The fusion protein of claim 33, in which a proline residueis located between the N-terminal of the polypeptide and the C-terminalof the cleavage peptide.
 43. The expression system of claim 25, whereinautolysis by the peptide encoded by sequence a) yields a polypeptidehaving an additional N-terminal proline residue and 0, 1 or a pluralityof further amino acid residues, and wherein any said additional aminoacid residue beyond said proline is removed by aminopeptidase P(3.4.11.9).
 44. The expression system of claim 43, wherein, aftercleavage up to the the proline residue, the proline residue is thenremoved by the action of proline iminopeptidase.
 45. The fusion proteinof claim 33, in which an alanine residue is located between theN-terminal of the polypeptide and the C-terminal of the cleavagepeptide.
 46. The expression system of claim 25, wherein autolysis by thepeptide encoded by sequence a) yields a polypeptide having an additionalN-terminal alanine residue and 0, 1 or a plurality of further amino acidresidues, and wherein any said additional amino acid residue beyond saidalanine is removed by aminopeptidase P (3.4.11.9), and the alanineresidue is removed by the action of alanine aminopeptidase (3.4.11.14).47. A polypeptide having the sequence given by residue numbers 4 to 437in sequence ID number 2 in the sequence listing, or a mutant or variantthereof.
 48. A polypeptide having the sequence given by residue numbers4 to 437 in sequence ID number 2 in the sequence listing.
 49. Apolynucleotide sequence encoding a polypeptide which comprises the aminoacid sequence of amino acid numbers 1 to 526 of sequence ID number 12,or which encodes a mutant or variant of said polypeptide, provided thatthe polypeptide encoded by the polynucleotide sequence is capable ofreducing dichloroindophenol and oxidized glutathione.
 50. Thepolynucleotide sequence of claim 49, which shares 55% sequence homology,or more, with amino acid numbers 1 to 526 of sequence ID number
 12. 51.The polynucleotide sequence of claim 49, which shares in excess of 70%sequence homology with amino acid numbers 1 to 526 of sequence ID number12.
 52. The polynucleotide sequence of claim 49, which shares in excessof 80% sequence homology with amino acid numbers 1 to 526 of sequence IDnumber
 12. 53. The polynucleotide sequence of claim 49, wherein thecoding sequence comprises the nucleotide sequence 70 to 1647 indicatedin sequence ID number
 11. 54. The polynucleotide sequence of claim 49,which encodes a polypeptide having the sequence −23 to 526 of sequenceID 12, or a mutant or variant thereof.
 55. The polypeptide encoded bythe polynucleotide sequence of claim
 49. 56. The polypeptide encoded bythe polynucleotide sequence of claim
 50. 57. The polypeptide encoded bythe polynucleotide sequence of claim
 51. 58. The polypeptide encoded bythe polynucleotide sequence of claim
 52. 59. The polypeptide encoded bythe polynucleotide sequence of claim
 53. 60. The polypeptide encoded bythe polynucleotide sequence of claim
 54. 61. A method for theprophylaxis or treatment of conditions caused by, or related to,oxidative stress, or any disease caused by activated oxygen, comprisingthe administration to a mammal in need thereof an effective, non-toxicdose of a peptide encoded by the polynucleotide sequence of claim 49.62. The polypeptide encoded by the polynucleotide sequence of claim 49,for use in the prophylaxis or therapy of arteriosclerosis, diabetes, orischemic disorders.
 63. A method for the prophylaxis or treatment ofarteriosclerosis, diabetes, ischemic disorders, edema, vascularhyperpermeability, inflammation, gastric mucosa disorders, acutepancreatitis, Crohn's disease, ulcerative colitis, liver disorders,Paraquat's disease, pulmonary emphysema, chemocarcinogenesis,carcinogenic metastasis, adult respiratory distress syndrome,disseminated intravascular coagulation, cataracts, prematureretinopathy, auto-immune diseases, porphyremia, hemolytic diseases,Mediterranean anemia, Parkinson's disease, Alzheimer's disease,epilepsy, ultraviolet radiation disorders, radioactive disorders,frostbite or burns, comprising the administration to a mammal in needthereof an effective, non-toxic dose of a peptide encoded by thepolynucleotide sequence of claim
 49. 64. A pharmaceutical compositioncomprising a pharmaceutically active amount of the peptide encoded bythe polynucleotide sequence of claim 49 together with a pharmaceuticallyacceptable carrier therefor.
 65. A vector containing the polynucleotidesequence of claim
 49. 66. An expression vector containing thepolynucleotide sequence of claim
 49. 67. A host transformed with thevector of claim
 65. 68. A host transformed with the vector of claim 66.69. An expression system comprising the host of claim
 68. 70. Thepolypeptide produced by the expression system of claim
 69. 71. Anantibody or an equivalent thereof, which specifically recognizes KM31-7protein, or which specifically recognizes a mutant or variant of KM31-7protein.
 72. The antibody of claim 71 which is a monoclonal antibody.73. The antibody of claim 71, wherein said antibody antigenicallyresembles a human antibody.
 74. An anti-idiotype antibody recognizingthe recognition site of the antibody of claim
 71. 75. The antibody ofclaim 71, as produced by the hybridoma designated MKM150-2 and depositedat the Fermentation Research Institute of the Agency of IndustrialScience and Technology, Japan, under the deposit number FERM BP-5086, oran antibody derived from a hybridoma obtained from the hybridomadesignated MKM150-2.
 76. The antibody of claim 71, for use in theisolation and purification of KM31-7 protein.
 77. A process for thepurification of KM31-7 protein comprising the use of the antibody ofclaim 71 to bind said protein.
 78. A hybridoma which expresses theantibody of claim 71 in culture.
 79. The hybridoma designated MKM150-2and deposited at the Fermentation Research Institute of the Agency ofIndustrial Science and Technology, Japan, under the deposit number FERMBP-5086.
 80. A polypeptide comprising the sequence given by residues 4to 437 in sequence ID number 2, or a mutant or variant thereof.
 81. DNAwhich hybridizes with DNA having the nucleotide sequence as given bynumbers 10 to 1311 in sequence ID number 1 in the sequence listing, andwherein the corresponding sense strand encodes a protein having proteaseactivity.
 82. The DNA according to claim 81, wherein the conditions ofhybridization use 6×SSC at 60 to 70° C.
 83. The expression system ofclaim 25, wherein the host is Escherichia coli.
 84. The expressionsystem of claim 69, wherein the host is Escherichia coli.
 85. DNA whichhybridizes with the polynucleotide sequence of claim 49, and wherein thecorresponding sense strand encodes a polypeptide having reducingactivity.
 86. The DNA of claim 85, wherein the conditions ofhybridization use 6×SSC at 60 to 70° C.
 87. The polynucleotide sequenceof claim 1, wherein the polypeptide encoded by the sequence of c) is apolypeptide which comprises the amino acid sequence of amino acidnumbers 1 to 526 of sequence ID number 12, or is a mutant or variant ofsaid polypeptide, provided that the polypeptide encoded by thepolynucleotide sequence is capable of reducing dichloroindophenol andoxidized glutathione.
 88. The fusion protein encoded by thepolynucleotide sequence of claim 87.